A method for detecting polycyclic aromatic hydrocarbons in water bodies by high performance liquid chromatography

Abstract

The application discloses a kind of water body polycyclic aromatic hydrocarbon's high performance liquid chromatography detection method, the present application relates to water quality pollution detection technical field, break polycyclic aromatic hydrocarbon detection relies on the industry solidification cognition of organic solvent, whole no organic reagent, through temperature-sensitive polymer pure water phase enrichment, whole water phase temperature-programmed chromatography separation, realize target accurate and efficient detection, the advantages of the present application are that: through the specific inclusion enrichment of temperature-sensitive type-cyclodextrin grafted supramolecular polymer pure water phase, combined with the integrated technical scheme of whole water phase high performance liquid chromatography separation, it breaks through the solidification technical cognition that polycyclic aromatic hydrocarbon enrichment, elution and chromatographic separation must rely on toxic organic solvent in the industry for a long time, completely eliminates the use of organic reagent in detection whole process, solve the occupational health exposure risk brought by detection personnel long-term contact carcinogenic organic solvent in traditional detection method.

Description

Technical Field

[0001] This invention relates to the field of water pollution detection technology, specifically a high-performance liquid chromatography method for the detection of polycyclic aromatic hydrocarbons in water. Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants composed of two or more aromatic rings. They possess strong carcinogenic, teratogenic, and mutagenic effects and are key toxic pollutants controlled in industrial wastewater treatment in industries such as chemical, coking, and pharmaceutical manufacturing. PAHs exhibit environmental persistence, are difficult to biodegrade, and are bioaccumulative. Conventional industrial wastewater treatment processes have limited removal capabilities, easily leading to excessive discharge of treated effluent and posing ecological risks to watershed aquatic environments. National standards in my country, such as the "Integrated Wastewater Discharge Standard" and the "Surface Water Environmental Quality Standard," as well as international control systems such as the US EPA and the EU Water Framework Directive, all include 16 common PAHs in the priority control pollutant list for water bodies, establishing strict emission limits. Therefore, accurate and efficient quantitative detection of PAHs in water bodies has become a core technological foundation for industrial wastewater treatment process development, water pollution source control, effluent compliance assessment, and watershed water pollution control. Existing high-performance liquid chromatography (HPLC) techniques for detecting polycyclic aromatic hydrocarbons (PAHs) in water primarily rely on liquid-liquid extraction or solid-phase extraction with organic reagents for target enrichment, followed by gradient elution of the organic mobile phase combined with fluorescence detection for qualitative and quantitative analysis. This approach, however, has significant limitations in practical water pollution treatment applications. Firstly, the entire detection process relies on toxic organic solvents such as methanol and acetonitrile, posing occupational health risks to personnel and generating large amounts of toxic wastewater that can cause secondary water pollution. Furthermore, the cumbersome procedures and high equipment and operational barriers make it unsuitable for rapid detection needs in industrial wastewater treatment sites, emergency water pollution response, and grassroots water pollution control stations. Secondly, multi-step organic phase operations can easily lead to the loss of PAHs, and gradient elution and wavelength switching can cause baseline drift and detection fluctuations, failing to accurately reflect the actual treatment effect of wastewater processes. This results in insufficient accuracy and stability of the detection results, hindering the precise control required for water pollution management. Therefore, we propose an HPLC method for detecting PAHs in water. Summary of the Invention

[0003] The purpose of this invention is to provide a high-performance liquid chromatography method for the detection of polycyclic aromatic hydrocarbons in water.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a high-performance liquid chromatography (HPLC) method for the detection of polycyclic aromatic hydrocarbons (PAHs) in water, the detection method comprising the following steps: Step 1: Take a water sample to be tested, filter it through a water-based polyethersulfone filter membrane with a pore size of 0.22-0.45μm to remove suspended particulate matter, algae and insoluble impurities, collect the clear water sample, seal it and store it in the dark. No organic reagents are involved in the whole process. Step 2: Add a temperature-sensitive agent to the clarified water sample. -Cyclodextrin grafted supramolecular polymers, vibrated at room temperature in the dark, via a temperature-sensitive method -Cyclodextrin grafted onto supramolecular polymers - The hydrophobic cavity of cyclodextrin enables the specific inclusion and capture of 16 preferentially controlled polycyclic aromatic hydrocarbons (PAHs) with 2-6 rings in water in a pure aqueous phase without the addition of organic extractants. Step 3: Heat the water sample system to a temperature-sensitive temperature. - Above the critical phase transition temperature of cyclodextrin-grafted supramolecular polymers, isothermal static incubation allows for the formation of temperature-sensitive polymers carrying 2-6 rings of 16 preferentially controlling polycyclic aromatic hydrocarbons. - cyclodextrin-grafted supramolecular polymers were quantitatively precipitated, and the supernatant was discarded after centrifugation to obtain a solid-phase enrichment material enriched with 16 preferentially controlled polycyclic aromatic hydrocarbons of 2-6 rings, without the participation of organic eluents throughout the process. Step 4: Add low-temperature ultrapure water to the solid enrichment material and make up the volume. Vortex mix to obtain a uniform injection suspension. No organic co-solvents are added throughout the process. Step 5: Inject the sample suspension into the high-performance liquid chromatography system, using... - Cyclodextrin-bonded phase chromatographic column, using ultrapure water as the sole mobile phase, achieves complete baseline separation of 16 preferentially controlled polycyclic aromatic hydrocarbons of 2-6 rings through programmed temperature rise, without the addition of organic mobile phase modifiers throughout the process; Step 6: Use a dual-channel fluorescence detector to simultaneously detect 16 priority control polycyclic aromatic hydrocarbons with 2-6 rings after separation using two sets of fixed wavelengths. Qualitative analysis is performed based on retention time, and no program wavelength switching is required throughout the process. Step 7: Establish a standard curve using the external standard method, and calculate the mass concentration of 16 priority controlled polycyclic aromatic hydrocarbons (PAHs) with 2-6 rings in the water sample using quantitative calculation formulas to complete the quantitative detection of all components.

[0005] As a further aspect of the present invention: In step one, the water sample is filtered under negative pressure using an aqueous polyethersulfone filter membrane at 15-28℃ to remove suspended particulate matter, algae, and insoluble impurities from the water. Before filtration, the filter membrane is pre-washed with ultrapure water. The volume ratio of ultrapure water used for pre-washing to the water sample to be filtered is 1:10-1:5. The clarified water sample after filtration is collected, sealed, and stored in a light-protected environment below 4℃ for later use. The water sample to be tested includes surface water, groundwater, domestic sewage, industrial wastewater, and seawater. No organic reagents are involved in this step.

[0006] As a further aspect of the present invention: In step two, the clarified water sample obtained in step one is taken, and a temperature-sensitive additive is added to the water sample at a material-to-liquid ratio of 0.5-2.0 g / L. -Cyclodextrin-grafted supramolecular polymers, the thermosensitive type - Cyclodextrin-grafted supramolecular polymers are based on N-isopropylacrylamide as the thermosensitive main chain monomer and monosubstituted methacrylates. -Cyclodextrin is a functional grafted monomer prepared by free radical solution polymerization, wherein N-isopropylacrylamide is esterified with monosubstituted methacrylate. - The molar ratio of cyclodextrins is 18:1-22:1, and the temperature-sensitive type The critical phase transition temperature of cyclodextrin-grafted supramolecular polymers is 30-35℃. - Cyclodextrin grafting rate ≥35%, under room temperature (15-28℃) and light-protected conditions, constant temperature shaking at 100-250 rpm for 5-20 minutes to achieve temperature-sensitive... - The cyclodextrin-grafted supramolecular polymer was completely and uniformly dispersed in the water sample, and the temperature-sensitive... -Cyclodextrin grafted onto supramolecular polymer molecules - The hydrophobic cavity of cyclodextrin specifically encapsulates 16 preferentially controlled polycyclic aromatic hydrocarbons (PAHs) of 2-6 rings in water, completing the molecular-level capture of the target analyte in a pure water system and obtaining a water sample system with the inclusion reaction completed. No organic extractants or inclusion aids are added throughout this step.

[0007] As a further aspect of the present invention: In step three, the water sample system that has undergone the inclusion reaction in step two is heated to a temperature higher than that of the thermosensitive type under sealed and light-protected conditions at a heating rate of 5-15℃ / min. - Cyclodextrin-grafted supramolecular polymers are kept at a constant temperature within the critical phase transition temperature range of 1-5℃ for 3-10 minutes to induce a thermosensitive polymer carrying 2-6 rings of 16 preferentially controlled polycyclic aromatic hydrocarbons. - Cyclodextrin grafted onto supramolecular polymers undergoes a hydrophobic phase transition, quantitatively precipitating from the water sample to form a solid-phase enriched material. Subsequently, it is centrifuged at 2000-5000 rpm for 2-5 minutes to allow the solid-phase enriched material to completely settle to the bottom of the container. All supernatant is discarded, yielding a solid-phase enriched material enriched with all 16 preferentially controlled polycyclic aromatic hydrocarbons of 2-6 rings. This step involves no organic eluents or concentrating reagents, completely avoiding the volatilization and loss of the target analytes during the transfer and concentration process.

[0008] As a further aspect of the present invention: in step four, the material enriched in step three is immediately added to the solid phase enrichment material, which has been pre-cooled to a temperature below that of a thermosensitive material. -Ultrapure water with a critical phase transition temperature of 5-10℃ for cyclodextrin-grafted supramolecular polymers is brought to a final volume of 0.5-2.0 mL. The mixture is then vortexed at 1500-3000 rpm for 20-60 seconds under the same low-temperature conditions to completely redisperse the solid-phase enrichment material in the ultrapure water, forming a homogeneous and stable injection suspension. No organic co-solvents or dispersants are added throughout this step, and the low-temperature environment is strictly controlled within temperature-sensitive limits. -Cyclodextrin-grafted supramolecular polymers below their critical phase transition temperature to avoid the preferential control of 16 types of 2-6 ring polycyclic aromatic hydrocarbons. -Cyclodextrin dissociates in the cavity.

[0009] As a further aspect of the present invention: in step five, the injection suspension obtained in step four is injected into a high-performance liquid chromatography system for chromatographic separation, using... - Cyclodextrin-bonded phase chromatographic column, column specifications 150-300mm × 4.6mm, packing particle size 3-10μm, using ultrapure water as the sole mobile phase, constant mobile phase flow rate 0.8-1.5mL / min, injection volume 5-50μL, column temperature controlled using programmed temperature ramp mode, specific temperature program as follows: initial column temperature set to 25-35℃, hold at 1-3min, then linearly ramp to 40-50℃ at 1-3℃ / min, hold at 2-4min, then ramp at 3-6℃ / min. The temperature was increased to 50-60℃ at a linear heating rate of ℃ / min and held at an isothermal position until all 16 preferentially controlled polycyclic aromatic hydrocarbons (PAHs) with 2-6 rings were eluted. The inclusion-dissociation equilibrium between PAHs with different ring numbers and the stationary phase of the chromatographic column was controlled by programmed temperature rise, achieving complete baseline separation of the 16 preferentially controlled PAHs with 2-6 rings with extremely wide polarity in a pure water mobile phase system. No organic mobile phase modifiers or gradient elution reagents were added throughout this step, completely abandoning the rigid logic of existing technologies that rely on organic phase to control the retention behavior of target substances.

[0010] As a further scheme of the present invention: in the step six, a dual-channel fluorescence detector equipped with a high performance liquid chromatography system is used to synchronously detect 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings after chromatographic separation completed in step five, and fixed detection wavelengths are set for the dual channels, wherein the excitation wavelength of the first detection channel is 260-280 nm and the emission wavelength is 330-350 nm, for quantitative response detection of 16 priority-controlled polycyclic aromatic hydrocarbon components with 2-6 rings of low-molecular-weight 2-3 rings, the excitation wavelength of the second detection channel is 290-305 nm and the emission wavelength is 420-440 nm, for quantitative response detection of 16 priority-controlled polycyclic aromatic hydrocarbon components with 2-6 rings of high-molecular-weight 4-6 rings, the data acquisition frequency of the detector is set to 5-20 Hz, qualitative judgment is carried out based on the retention time of chromatographic peaks of the 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings, and the relative deviation between the actually measured retention time of the 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings and the retention time of the corresponding standard substance is ≤±3%; no programmed wavelength switching is required throughout this step, which avoids baseline drift and detection response fluctuation, and ensures the accuracy and stability of qualitative results.

[0011] As a further scheme of the present invention: in the step seven, an external standard method is used to quantitatively calculate 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings in a water sample, standard substances of 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings are pre-treated according to the same process of steps one to six, and a standard curve of the mass concentration of 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings and the corresponding chromatographic peak area is established, and the linear correlation coefficient R of the standard curve 2 ≥ 0.995, and the mass concentration of each 16 priority-controlled polycyclic aromatic hydrocarbon component with 2-6 rings in the water sample to be detected is calculated according to the following formula: ; wherein, is the mass concentration of 16 priority-controlled polycyclic aromatic hydrocarbon components with 2-6 rings in the water sample to be detected, with the unit of ng / L, is the mass concentration of 16 priority-controlled polycyclic aromatic hydrocarbon components with 2-6 rings in the injection suspension calculated according to the standard curve, with the unit of ng / mL, is the constant volume of the injection suspension in step four, with the unit of mL, is the sampling volume of the water sample to be detected used for enrichment treatment in step two, with the unit of L, methodological quality control is completed at the same time, the relative standard deviation of the detection results of parallel samples is ≤3%, the recovery rate of standard addition is controlled between 98% and 102%, and the full-component quantitative detection results of 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings in the water sample are output.

[0012] By adopting the above technical scheme, compared with the prior art, the beneficial effect of the present invention is that: 1. The present invention achieves, through the temperature-sensitive This integrated technology, combining the specific inclusion enrichment of cyclodextrin-grafted supramolecular polymers in pure aqueous phase with all-aqueous high-performance liquid chromatography (HPLC), breaks through the long-standing industry perception that the enrichment, elution, and chromatographic separation of polycyclic aromatic hydrocarbons (PAHs) must rely on toxic organic solvents. It completely eliminates the use of organic reagents from the entire detection process. This solution addresses the occupational health risks posed by long-term exposure of testing personnel to carcinogenic organic solvents in traditional methods. It also avoids the subsequent disposal costs and secondary water pollution problems caused by the generation of large amounts of toxic waste during the testing process. Furthermore, it significantly simplifies the testing process, reducing the entire single-sample testing cycle from over 4 hours in traditional methods to less than 15 minutes. This lowers the equipment and cost barriers to entry, making the method perfectly adaptable to various applications such as on-site screening of industrial wastewater treatment, emergency response to water pollution, daily monitoring at grassroots water pollution control stations, and precise laboratory testing. It achieves green, low-threshold, and standardized detection of PAHs in water bodies, adaptable to the entire water pollution treatment process. 2. This invention is achieved through... - The hydrophobic cavity of cyclodextrin enables molecular-level specific inclusion of polycyclic aromatic hydrocarbons (PAHs) to achieve in-situ enrichment of the target analytes in pure aqueous phase. Combined with temperature-programmed baseline separation technology in aqueous chromatography, this method completely avoids the loss of PAHs caused by multiple steps of organic reagent extraction, transfer, and concentration in traditional detection methods. This approach eliminates the organic phase gradient elution and programmed wavelength switching modes that easily cause detection fluctuations in traditional methods, reducing baseline drift and response fluctuations during the detection process. It effectively improves the accuracy and stability of qualitative and quantitative detection of PAHs in water, allowing the detection results to more realistically reflect the actual occurrence state of PAHs in water, and providing reliable technical support for pollution control and risk assessment of persistent organic pollutants in the aquatic environment.

[0013] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the method steps in an embodiment of the present invention; Figure 2 This is a schematic diagram of the sample pretreatment process in an embodiment of the present invention; Figure 3 This is a schematic diagram of the chromatographic separation and detection process in an embodiment of the present invention. Detailed Implementation

[0015] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0016] Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0017] Please see the appendix Figure 1 -Appendix Figure 3 This invention provides a high-performance liquid chromatography (HPLC) method for the detection of polycyclic aromatic hydrocarbons (PAHs) in water. The detection method includes the following steps: Step 1: Take a water sample to be tested, filter it through a water-based polyethersulfone filter membrane with a pore size of 0.22-0.45μm to remove suspended particulate matter, algae and insoluble impurities, collect the clear water sample, seal it and store it in the dark. No organic reagents are involved in the whole process. Step 2: Add a temperature-sensitive agent to the clarified water sample. -Cyclodextrin grafted supramolecular polymers, vibrated at room temperature in the dark, via a temperature-sensitive method -Cyclodextrin grafted onto supramolecular polymers - The hydrophobic cavity of cyclodextrin enables the specific inclusion and capture of 16 preferentially controlled polycyclic aromatic hydrocarbons (PAHs) with 2-6 rings in water in a pure aqueous phase without the addition of organic extractants. Step 3: Heat the water sample system to a temperature-sensitive temperature. - Above the critical phase transition temperature of cyclodextrin-grafted supramolecular polymers, isothermal static incubation allows for the formation of temperature-sensitive polymers carrying 2-6 rings of 16 preferentially controlling polycyclic aromatic hydrocarbons. - cyclodextrin-grafted supramolecular polymers were quantitatively precipitated, and the supernatant was discarded after centrifugation to obtain a solid-phase enrichment material enriched with 16 preferentially controlled polycyclic aromatic hydrocarbons of 2-6 rings, without the participation of organic eluents throughout the process. Step 4: Add low-temperature ultrapure water to the solid enrichment material and make up the volume. Vortex mix to obtain a uniform injection suspension. No organic co-solvents are added throughout the process. Step 5: Inject the sample suspension into the high-performance liquid chromatography system, using... - Cyclodextrin-bonded phase chromatographic column, using ultrapure water as the sole mobile phase, achieves complete baseline separation of 16 preferentially controlled polycyclic aromatic hydrocarbons of 2-6 rings through programmed temperature rise, without the addition of organic mobile phase modifiers throughout the process; Step 6: Use a dual-channel fluorescence detector to simultaneously detect 16 priority control polycyclic aromatic hydrocarbons with 2-6 rings after separation using two sets of fixed wavelengths. Qualitative analysis is performed based on retention time, and no program wavelength switching is required throughout the process. Step 7: Establish a standard curve using the external standard method, and calculate the mass concentration of 16 priority controlled polycyclic aromatic hydrocarbons (PAHs) with 2-6 rings in the water sample using quantitative calculation formulas to complete the quantitative detection of all components.

[0018] Specifically: Thermosensitive β-cyclodextrin-grafted supramolecular polymers are prepared via free radical solution polymerization, with the following specific steps: S1. Take a 250mL three-necked reaction flask and continuously purge it with high-purity nitrogen for 30 minutes to completely purge the air from the flask. Add 100mL of ultrapure water with a resistivity of 18.2MΩ・cm to the flask, then add 2.26g of N-isopropylacrylamide (thermosensitive main chain monomer) and 1.13g of monosubstituted methacrylated β-cyclodextrin (functional grafted monomer), with a molar ratio of 20:1. Stir at room temperature until the monomers are completely dissolved to obtain a clear and transparent monomer mixed solution. S2. Add 0.045g of initiator ammonium persulfate to the monomer mixture solution, stir until completely dissolved, add 60μL of catalyst tetramethylethylenediamine, continuously purge with nitrogen for protection, and react in a sealed environment at a constant temperature of 25℃ for 6h to obtain a milky white polymerization reaction solution. S3. The polymerization reaction solution is placed into a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyzed with ultrapure water for 72 hours. Fresh ultrapure water is replaced every 12 hours to completely remove unreacted monomers and small molecule impurities. S4. The purified product by dialysis was placed in a freeze dryer at -50℃ and freeze-dried for 48 hours to obtain a white powdery thermosensitive β-cyclodextrin-grafted supramolecular polymer. The critical phase transition temperature of the polymer was found to be 32℃ and the β-cyclodextrin grafting rate was 42%, which met the performance requirements of the technical solution of this invention. Example

[0019] This embodiment uses surface water from a river as the water sample to be tested, and detects 16 preferentially controlled polycyclic aromatic hydrocarbons in the water. The specific steps are as follows: Step 1: Take 1000mL of surface water sample to be tested. At room temperature of 25℃, use a water-based polyethersulfone filter membrane with a pore size of 0.22μm to filter the water sample under negative pressure to remove suspended particulate matter, algae and insoluble impurities from the water. Before filtration, the filter membrane was pre-washed with 100 mL of ultrapure water. The volume ratio of the ultrapure water used for pre-washing to the water sample to be filtered was 1:10. Collect the filtered clear water sample, seal it, and store it at 4°C in a dark environment for later use. No organic reagents are involved in this step.

[0020] Step 2: Take 500 mL of the clarified water sample obtained in Step 1, and add 0.5 g of the temperature-sensitive material prepared above to the water sample at a material-to-liquid ratio of 1.0 g / L. - Cyclodextrin-grafted supramolecular polymers were subjected to isothermal shaking at 200 rpm for 10 minutes at 25°C in the dark to ensure complete and uniform dispersion of the polymer in the water sample. This dispersion was achieved through the molecular structure of the polymer. - The hydrophobic cavity of cyclodextrin specifically encapsulates 16 preferentially controlled polycyclic aromatic hydrocarbons (PAHs) of 2-6 rings in water, completing the molecular-level capture of the target analyte in a pure water system and obtaining a water sample system with the inclusion reaction completed. No organic extractants or inclusion aids are added throughout this step.

[0021] Step 3: Under closed and light-proof conditions, heat the water sample system that has completed the inclusion reaction in Step 2 to 34°C at a heating rate of 10°C / min. This temperature is 2°C higher than the critical phase transition temperature of the polymer. Keep it at this temperature for 5 minutes to allow the polymer carrying the target polycyclic aromatic hydrocarbon to undergo a hydrophobic phase transition and quantitatively precipitate from the water sample to form a solid enrichment material. The solid-phase enriched material was then centrifuged at 3000 rpm for 3 minutes to allow it to settle completely to the bottom of the container. All supernatant was discarded, resulting in a solid-phase enriched material containing all 16 preferentially controlled polycyclic aromatic hydrocarbons. No organic eluent or concentrating reagent was involved in this step.

[0022] Step 4: Immediately add pre-cooled ultrapure water to the solid enriched material obtained in Step 3. This temperature is 7°C lower than the critical phase transition temperature of the polymer. Make up the volume to 1.0 mL and vortex mix at 2000 rpm for 30 seconds at a low temperature of 25°C to completely redisperse the solid enriched material in the low-temperature ultrapure water and form a uniform and stable injection suspension. No organic co-solvents or dispersants are added in this step.

[0023] Step 5: Inject the sample suspension obtained in Step 4 into a high-performance liquid chromatography (HPLC) system for chromatographic separation. - Cyclodextrin-bonded phase chromatographic column, column size 250mm×4.6mm, packing particle size 5μm; Ultrapure water was used as the sole mobile phase, with a constant flow rate of 1.0 mL / min and an injection volume of 20 μL. The column temperature was controlled using a programmed temperature ramping mode. The specific temperature ramping program was as follows: the initial column temperature was set to 30℃ and held for 2 min. Then, the temperature was increased to 45℃ at a linear ramping rate of 2℃ / min and held for 3 min. Finally, the temperature was increased to 55℃ at a linear ramping rate of 4℃ / min and held until all 16 polycyclic aromatic hydrocarbons had eluted. The total separation time was 15 min. By controlling the inclusion-dissociation equilibrium between polycyclic aromatic hydrocarbons (PAHs) with different ring numbers and the stationary phase of the chromatographic column through programmed temperature rise, complete baseline separation of 16 PAHs with a wide range of polarities was achieved in a pure water mobile phase system. No organic mobile phase modifiers or gradient elution reagents were added throughout this step.

[0024] Step 6: A dual-channel fluorescence detector equipped with a high performance liquid chromatography system is used to perform simultaneous detection on the 16 polycyclic aromatic hydrocarbons that have completed chromatographic separation in Step 5. Fixed detection wavelengths are set for the dual channels, wherein the excitation wavelength of the first detection channel is 275 nm and the emission wavelength is 340 nm, which is used for quantitative response detection of 2-3 ring low-molecular-weight polycyclic aromatic hydrocarbon components; the excitation wavelength of the second detection channel is 295 nm and the emission wavelength is 430 nm, which is used for quantitative response detection of 4-6 ring high-molecular-weight polycyclic aromatic hydrocarbon components; The data acquisition frequency of the detector is set to 10 Hz. Qualitative determination is performed based on the chromatographic peak retention times of the 16 polycyclic aromatic hydrocarbons, and the relative deviation between the actually measured retention time of the target substance and the retention time of the corresponding standard substance is ≤ ±2%. No programmed wavelength switching is required throughout this step.

[0025] Step 7: An external standard method is used for quantitative calculation of 16 polycyclic aromatic hydrocarbons in water samples. A mixed standard substance of 16 polycyclic aromatic hydrocarbons is pre-treated according to the same process from Step 1 to Step 6 to establish a standard curve between the mass concentration of each of the 16 polycyclic aromatic hydrocarbons and the corresponding chromatographic peak area. The linear correlation coefficient R of the standard curve 2 ≥ 0.999; Calculate the mass concentration of each polycyclic aromatic hydrocarbon component in the water sample to be tested according to the following formula: ; In the formula: is the mass concentration of the target polycyclic aromatic hydrocarbon component in the water sample to be tested, with a unit of ng / L, is the mass concentration of the target polycyclic aromatic hydrocarbon component in the injected suspension calculated according to the standard curve, with a unit of ng / mL, is the constant volume of the injected suspension in Step 4, with a unit of mL, is the sampling volume of the water sample to be tested used for enrichment in Step 2, with a unit of L; Methodological quality control is completed at the same time, the relative standard deviation of detection results of parallel samples is ≤ 2%, and finally the quantitative detection results of all components of 16 priority-controlled polycyclic aromatic hydrocarbons in the water sample are output.

[0026] After detection, 5 polycyclic aromatic hydrocarbon components including naphthalene, fluorene, phenanthrene, fluoranthene and pyrene are detected in the surface water sample in this example, their mass concentrations are 12.5 ng / L, 8.2 ng / L, 15.6 ng / L, 22.3 ng / L and 18.7 ng / L respectively, and the remaining 11 polycyclic aromatic hydrocarbon components are not detected. Example

[0027] In this example, industrial wastewater from a chemical industrial park is taken as the water sample to be tested, and detection of 16 priority-controlled polycyclic aromatic hydrocarbons in the water is carried out. The specific steps are as follows: Step 1: Take 1000 mL of the industrial wastewater sample to be tested. At room temperature of 25℃, use a water-based polyethersulfone filter membrane with a pore size of 0.22 μm to filter the water sample under negative pressure to remove suspended particulate matter and insoluble impurities from the water. The filter membrane was pre-washed with 100mL of ultrapure water before filtration. Collect the filtered clear water sample, seal it, and store it at 4°C in a dark environment for later use. No organic reagents are involved in this step.

[0028] Step 2: Take 500 mL of the clarified water sample obtained in Step 1, and add 0.75 g of the temperature-sensitive material prepared above to the water sample at a material-to-liquid ratio of 1.5 g / L. -Cyclodextrin grafted onto supramolecular polymers was subjected to constant temperature oscillation at 220 rpm for 15 min at room temperature (25°C) in the dark to achieve molecular-level capture of the target substance in a pure water system, resulting in a water sample system with complete inclusion reaction. No organic extractants or inclusion aids were added during this step.

[0029] Step 3: The water sample system that underwent inclusion reaction in Step 2 was heated to 34°C at a heating rate of 10°C / min under sealed and light-protected conditions, kept at the same temperature for 8 minutes, and then centrifuged at 4000 rpm for 4 minutes. All supernatant was discarded to obtain solid-phase enrichment material enriched with all 16 preferentially controlled polycyclic aromatic hydrocarbons. No organic eluent or concentrating reagent was involved in this step.

[0030] Step 4: Immediately add pre-cooled ultrapure water to the solid enrichment material obtained in Step 3 and bring the volume to 1.0 mL. Vortex mix at 2500 rpm for 45 s at a low temperature of 25°C to form a uniform and stable injection suspension. No organic co-solvents or dispersants are added in this step.

[0031] Step 5: The chromatographic separation conditions are exactly the same as in Example 1, with ultrapure water as the only mobile phase. Complete baseline separation of 16 polycyclic aromatic hydrocarbons is achieved through programmed temperature rise, without the addition of any organic mobile phase modifier.

[0032] Step 6: The fluorescence detection conditions are exactly the same as in Example 1. Dual-channel fixed wavelength synchronous detection is used, and no program wavelength switching is required throughout the process. Qualitative analysis is performed by retention time.

[0033] Step 7: Quantitative calculation is performed using the external standard method. The standard curve establishment method and quantitative calculation formula are completely consistent with those in Example 1. After completing the methodological quality control, the quantitative detection results of all components of 16 preferentially controlled polycyclic aromatic hydrocarbons in the water sample are output. The thermosensitive β-cyclodextrin grafted supramolecular polymer used in Examples 1 and 2 has a molar ratio of N-isopropylacrylamide to monosubstituted methacrylated β-cyclodextrin of 20:1, a critical phase transition temperature of 32℃, and a β-cyclodextrin grafting rate of 42%.

[0034] The test results showed that the industrial wastewater sample in this embodiment contained nine polycyclic aromatic hydrocarbons (PAHs), namely naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, and α, with a mass concentration range of 15.2-186.4 ng / L. The other seven PAHs were not detected.

[0035] Comparative Example This comparative example uses the method in "Determination of Polycyclic Aromatic Hydrocarbons in Water by Liquid-Liquid Extraction High Performance Liquid Chromatography" (HJ478-2009) to simultaneously detect surface water samples identical to those in Example 1 and industrial wastewater samples identical to those in Example 2. The specific steps shall be carried out in accordance with national standard methods: Using a hexane-dichloromethane mixed solvent as the extractant, anhydrous sodium sulfate was used for dehydration. After rotary evaporation and concentration, the solution was diluted to volume with methanol. A C18 column was used with gradient elution using methanol-water as the mobile phase, combined with programmed wavelength switching fluorescence detection, and external standard method was used for quantification.

[0036] Spiked recovery experiments were simultaneously conducted on the corresponding water samples from Examples 1, 2, and Comparative Example 1. The spiked concentration for surface water was 50 ng / L, and the spiked concentration for industrial wastewater was 100 ng / L. Each water sample was measured in parallel six times. The comparison results of the core detection indicators of the two methods are shown in the table below:

[0037] This section provides a systematic methodological validation of the detection method of the present invention. The validation includes linear range, detection limit, precision, and accuracy. The specific results are as follows: A series of standard solutions of 16 polycyclic aromatic hydrocarbons (PAHs) were prepared with mass concentration gradients of 1.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 50.0 ng / mL, 100.0 ng / mL, and 200.0 ng / mL. These solutions were processed according to steps one through six of the method of this invention. A standard curve was plotted with mass concentration on the x-axis and the corresponding peak area on the y-axis. The results showed that the 16 PAHs exhibited good linearity in the range of 1.0–200.0 ng / mL, and the linear correlation coefficients (R²) of the standard curves were all ≥0.999, meeting the requirements for quantitative detection.

[0038] The method detection limit and quantitation limit were determined according to the requirements of the "Technical Guidelines for the Formulation and Revision of Environmental Monitoring Analytical Methods Standards" (HJ168-2020). Seven parallel determinations were performed on a blank spiked water sample with a spike concentration of 2.0 ng / L. The standard deviation (SD) of the determination results was calculated. The method detection limit was calculated, and the method quantitation limit was set at four times the detection limit. Results showed that the method of this invention has detection limits of 0.5-2.0 ng / L and quantitation limits of 2.0-8.0 ng / L for 16 polycyclic aromatic hydrocarbons, meeting the requirements for the detection of trace amounts of polycyclic aromatic hydrocarbons in surface water, groundwater, drinking water, and other water bodies.

[0039] For precision verification, spiked water samples at three concentration levels (10 ng / L, 50 ng / L, and 200 ng / L) were used. Six parallel determinations were performed according to the method of this invention, and the relative standard deviation (RSD) of the results was calculated. The results showed that at the three concentration levels, the intra-day RSD of the determination results for 16 polycyclic aromatic hydrocarbons was 0.8%–2.1%, and the inter-day RSD was 1.2%–3.5%, indicating that this method has excellent precision and stability.

[0040] Accuracy verification was performed using spiked recovery experiments. Spiked recovery experiments were conducted on water samples from four substrates: surface water, groundwater, industrial wastewater, and seawater. The spike concentration was 50 ng / L, and each substrate was measured in parallel six times. The results showed that the recoveries of 16 polycyclic aromatic hydrocarbons (PAHs) in the four substrates ranged from 97.5% to 102.3%, with a relative standard deviation (RSD) ≤ 2.5%. This indicates that the method has excellent accuracy and resistance to substrate interference, and is suitable for the detection needs of various water substrates.

[0041] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Any variations and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, any modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention, without departing from the scope of the invention, fall within the protection scope defined by the claims of the present invention.

Claims

1. A high-performance liquid chromatography method for the detection of polycyclic aromatic hydrocarbons in water, characterized in that, Said detection method comprises the following steps: Step 1: taking a water sample to be detected, filtering the water sample through an aqueous polyethersulfone filter membrane with a pore size of 0.22-0.45 μm to remove suspended particulate matters, algae and insoluble impurities, collecting the clarified water sample and storing the same in a sealed and light-proof condition; Step 2: Add a temperature-sensitive agent to the clarified water sample. -Cyclodextrin grafted onto supramolecular polymers, shaken at room temperature in the dark; Step 3: Heat the water sample system to a temperature-sensitive temperature. - After grafting supramolecular polymers with cyclodextrin above the critical phase transition temperature, the mixture is kept at a constant temperature and centrifuged, then the supernatant is discarded to obtain a solid-phase enrichment material enriched with 16 preferentially controlled polycyclic aromatic hydrocarbons of 2-6 rings. Step 4: adding low-temperature ultrapure water to the solid-phase enrichment material to a constant volume, and performing vortex mixing to prepare a uniform sampling suspension; Step 5: Inject the sample suspension into the high-performance liquid chromatography system, using... - Cyclodextrin-bonded phase chromatographic column, using ultrapure water as the sole mobile phase, to achieve complete baseline separation of 16 preferentially controlled polycyclic aromatic hydrocarbons of 2-6 rings by temperature programmed rise; Step 6: synchronously detecting 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings after separation by using a dual-channel fluorescence detector with two groups of fixed wavelengths, and conducting qualitative analysis through retention time; Step 7: establishing a standard curve by an external standard method, calculating the mass concentration of 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings in the water sample through a quantitative calculation formula, and completing the quantitative detection of all components.

2. The high-performance liquid chromatography method for detecting polycyclic aromatic hydrocarbons in water according to claim 1, characterized in that, In said step 1, the water sample is subjected to negative pressure filtration through the aqueous polyethersulfone filter membrane at a temperature of 15-28°C to remove suspended particulate matters, algae and insoluble impurities in the water; before filtration, the filter membrane is pre-rinsed with ultrapure water, wherein the volume ratio of the ultrapure water used for pre-rinsing to the water sample to be filtered is 1:10-1:5; the filtered clarified water sample is collected, sealed and stored for later use in a light-proof environment below 4°C; and the water sample to be detected comprises surface water, groundwater, domestic sewage, industrial wastewater and seawater.

3. The high-performance liquid chromatography method for detecting polycyclic aromatic hydrocarbons in water according to claim 1, characterized in that, In step two, the clarified water sample obtained in step one is taken, and a temperature-sensitive additive is added to the water sample at a material-to-liquid ratio of 0.5-2.0 g / L. -Cyclodextrin-grafted supramolecular polymers, the thermosensitive type - Cyclodextrin-grafted supramolecular polymers are based on N-isopropylacrylamide as the thermosensitive main chain monomer and monosubstituted methacrylates. -Cyclodextrin is a functional grafted monomer prepared by free radical solution polymerization, wherein N-isopropylacrylamide is esterified with monosubstituted methacrylate. - The molar ratio of cyclodextrins is 18:1-22:1, and the temperature-sensitive type The critical phase transition temperature of cyclodextrin-grafted supramolecular polymers is 30-35℃. - With a cyclodextrin grafting rate ≥35%, the target substance was captured at the molecular level in a pure water system by constant temperature oscillation at 100-250 rpm for 5-20 min at room temperature (15-28℃) in the dark, thus obtaining a water sample system that has completed the inclusion reaction.

4. The high-performance liquid chromatography method for detecting polycyclic aromatic hydrocarbons in water according to claim 1, characterized in that, In step three, the water sample system that underwent the inclusion reaction in step two is heated to a temperature higher than that of the thermosensitive type under sealed, light-protected conditions at a heating rate of 5-15℃ / min. - The critical phase transition temperature of the cyclodextrin-grafted supramolecular polymer is within the range of 1-5℃. After being kept at a constant temperature for 3-10 minutes, a solid-phase enriched material is quantitatively precipitated from the water sample. Then, it is centrifuged at 2000-5000 rpm for 2-5 minutes to allow the solid-phase enriched material to completely settle to the bottom of the container. All the supernatant is discarded to obtain a solid-phase enriched material enriched with all 16 preferentially controlled polycyclic aromatic hydrocarbons of 2-6 rings.

5. The high-performance liquid chromatography method for detecting polycyclic aromatic hydrocarbons in water according to claim 1, characterized in that, In step four, the material enriched in step three is immediately added to the solid phase, which has been pre-cooled to a temperature below that of the thermosensitive type. -Ultrapure water with a critical phase transition temperature of 5-10℃ for cyclodextrin-grafted supramolecular polymers is brought to a final volume of 0.5-2.0 mL and vortexed at 1500-3000 rpm for 20-60 s under the same low-temperature conditions to form a uniform and stable injection suspension.

6. The high-performance liquid chromatography method for detecting polycyclic aromatic hydrocarbons in water according to claim 1, characterized in that, In step five, the injection suspension obtained in step four is injected into a high-performance liquid chromatography system for chromatographic separation. - Cyclodextrin-bonded phase chromatographic column, column specifications 150-300mm×4.6mm, packing particle size 3-10μm, ultrapure water as the sole mobile phase, constant mobile phase flow rate 0.8-1.5mL / min, injection volume 5-50μL, column temperature controlled by programmed temperature ramping mode, specific temperature ramping program is as follows: initial column temperature set to 25-35℃, hold at temperature for 1-3min, then ramp to 40-50℃ at a linear temperature ramping rate of 1-3℃ / min, hold at temperature for 2-4min, then ramp to 50-60℃ at a linear temperature ramping rate of 3-6℃ / min, hold at temperature until all 2-6 ring 16 preferentially controlled polycyclic aromatic hydrocarbons have eluted.

7. The high-performance liquid chromatography method for detecting polycyclic aromatic hydrocarbons in water according to claim 1, characterized in that, In said step 6, the dual-channel fluorescence detector equipped with a high-performance liquid chromatography system is used to synchronously detect 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings that have completed chromatographic separation in step 5, and fixed detection wavelengths for two channels are set: the first detection channel has an excitation wavelength of 260-280 nm and an emission wavelength of 330-350 nm, and is used for quantitative response detection of 2-3 ring low-molecular-weight components among the 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings; the second detection channel has an excitation wavelength of 290-305 nm and an emission wavelength of 420-440 nm, and is used for quantitative response detection of 4-6 ring high-molecular-weight components among the 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings; the data acquisition frequency of the detector is set to 5-20 Hz; qualitative determination is performed based on the chromatographic peak retention time of the 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings, and the relative deviation between the actually measured retention time of the 16 priority-controlled polycyclic aromatic hydrocarbons with 2-6 rings and the retention time of the corresponding standard product is ≤±3%.

8. The high-performance liquid chromatography method for detecting polycyclic aromatic hydrocarbons in water according to claim 1, characterized in that, In step seven, the external standard method is used to quantitatively calculate the 16 preferentially controlled polycyclic aromatic hydrocarbons (PAHs) of 2-6 rings in the water sample. Standards for these 16 PHAs are pre-processed using the same procedures as steps one through six to establish a standard curve relating the mass concentration of the 16 PHAs to their corresponding chromatographic peak areas. The linear correlation coefficient R of the standard curve is... 2 For a concentration ≥0.995, calculate the mass concentration of each of the 16 preferentially controlled polycyclic aromatic hydrocarbon components (PAHs) with 2-6 rings in the water sample to be tested using the following formula: ； in, The concentration of 16 preferentially controlled polycyclic aromatic hydrocarbon components (PAHs) of rings 2-6 in the water sample to be tested is given in ng / L. The concentrations of the 16 preferentially controlled polycyclic aromatic hydrocarbon components (2-6 rings) in the injected suspension, calculated based on the standard curve, are expressed in ng / mL. This refers to the final volume of the injection suspension in step four, in mL. This represents the sampling volume of the water sample to be tested used for enrichment treatment in step two, in L. The output is the quantitative detection results of all components of 16 preferentially controlled polycyclic aromatic hydrocarbons (PAHs) with 2-6 rings in the water sample.

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