Sample pretreatment method for constructing iron-bound organic carbon molecule fingerprints in sediments

Abstract

The invention relates to the technical field of environmental geochemistry research, in particular to a sample pretreatment method for constructing iron-bound organic carbon molecule fingerprints in sediments. The invention provides a sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments. Iron-bound organic carbon is extracted from the sediments through water extraction, acid extraction and near-neutral reduction extraction in sequence according to a binding force sequence from weak to strong and from non-destruction to selective destruction. According to the method, Fe-OC sub-fractions controlled by different acting forces and mineral phases can be distinguished, meanwhile, extracted samples are desalted through unified solid-phase extraction, molecular fingerprint characterization of a fluorescence three-dimensional spectrum (EEM) and a high-resolution mass spectrum (HRMS) can be compatible, and data mutual identification and molecular fingerprint acquisition of the EEM and the HRMS on the same sample are achieved.

Description

Technical Field

[0001] This invention relates to the field of environmental geochemistry research technology, and in particular to a sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments. Background Technology

[0002] Iron-bound organic carbon (Fe-OC) is widely present in coastal or estuarine sediments and is an important form of organic carbon coupled with iron oxide cycling for long-term preservation. Accurate separation and characterization of Fe-OC can help elucidate the carbon burial mechanism in the land-sea transition zone, the impact of iron reduction / re-oxidation processes on organic matter preservation, and the interaction between dissolved organic matter (DOM) and metal ions.

[0003] The combined use of three-dimensional fluorescence spectroscopy (EEM) and high-resolution mass spectrometry (HRMS, such as FT-ICR MS) provides an irreplaceable and comprehensive perspective for studying iron-bound organic carbon in sediments, moving from a "collective portrait" to a "molecular identity card." This advances the study of iron-bound organic carbon from a "black box" or "collective statistics" level to a level of "molecular mechanism analysis." It allows researchers not only to know how much carbon iron "locks in," but also which specific molecules it locks in, and how these molecules will traverse under environmental perturbations. This is revolutionary for accurately assessing the stability of carbon sequestration, predicting responses to climate change, and understanding the microscopic mechanisms of carbon-iron co-evolution in Earth's history.

[0004] The ultimate goal of combining EEM spectroscopy and HRMS is to achieve direct molecular correlation and mutual verification of data. This requires that the extracted samples be compatible with both three-dimensional fluorescence spectroscopy and high-resolution mass spectrometry. For example, an intensity change of a "humic acid-like C peak" is observed in the EEM spectrum. In homologous split samples (i.e., one sample is split into two parts for EEM and HRMS testing respectively), it can be confirmed that changes in molecular composition detected by HRMS (such as an increase or decrease in the number of polyphenols and highly aromatic molecules) necessarily and directly lead to the change in this fluorescence peak. However, in samples obtained by separate processing, this correlation can only be inferred because there is a possibility that the humic acid molecules in the EEM sample are extracted more or less thoroughly, resulting in a signal mismatch with the HRMS sample.

[0005] Meanwhile, the dissolved organic carbon in the two extracts obtained by separate processing may differ, increasing the complexity and uncertainty of normalization. Summary of the Invention

[0006] To achieve the ultimate goal of combining EEM and HRMS, the present invention aims to provide a sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments. Iron-bound organic carbon samples obtained using the method disclosed in this invention can be directly used for EEM and HRMS detection.

[0007] This invention discloses a sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments, comprising the following steps:

[0008] S1: Mix the sediment with water and stir until all Fe-OC or DOM that can be leached by water are dissolved; centrifuge to obtain supernatant A and solid sediment A. S2: Mix the solid sludge A with hydrochloric acid aqueous solution, stir until the reaction is complete, centrifuge, and obtain supernatant B and solid sludge B; S3: Mix the solid sludge B with an aqueous solution of sodium bicarbonate and sodium dithionite, adjust the pH to 6.5-7.5 using a pH adjuster, stir until the reaction is complete, centrifuge, and obtain the supernatant C and residue; S4: Filter the supernatant A, supernatant B and supernatant C; mix them evenly, add a pH adjuster to adjust the pH of the mixture to 1.5-2.5 to obtain an acidified mixture; S5: The acidified mixture is subjected to solid-phase extraction for desalting and elution to obtain an eluent; S6: Concentrate the eluent by nitrogen blowing to obtain a test sample; divide the test sample into two parts and use them for fluorescence three-dimensional spectroscopy analysis (to obtain a fluorescent molecular fingerprint) and high-resolution mass spectrometry analysis (to obtain a mass spectrometry molecular fingerprint), respectively.

[0009] Molecular fingerprinting refers to obtaining comparable, traceable, and classifiable fluorescence and mass spectrometry fingerprints by taking raw data from the same sample and analyzing them separately using fluorescence three-dimensional spectroscopy (EEM) and high-resolution mass spectrometry (HRMS) under a unified processing procedure.

[0010] Fluorescent molecular fingerprint (EEM fingerprint) refers to a set of stable characteristic parameters obtained by normalizing (by DOC or total fluorescence) the three-dimensional fluorescence matrix after blank subtraction, Rayleigh / Raman removal, and internal filtration effect (IFE) correction. These parameters characterize the chromophore composition and structural information of a sample. Typical features include PARAFAC component scores (e.g., humic, protein / microbial components), the Ex / Em position and relative intensity of the main peak, peak ratios such as A / C and C / T, and optical indices such as FI, HIX, and BIX. This fingerprint reflects Fe-OC related aromaticity / humification degree, microbial origin characteristics, and the modulation of fluorescence by metal complexes, exhibiting sample specificity and reproducibility.

[0011] Mass spectrometry molecular fingerprints (HRMS fingerprints) refer to the set of molecular composition and structural features obtained by statistically analyzing high-resolution mass spectrometry (FT-ICR or Orbitrap) data using a uniform threshold and grid, after mass calibration, redundancy removal of isotopes / adducts, and molecular formula assignment. Typical features include elemental categories and their relative abundances (CHO, CHON, CHOS, CHOP, etc.), Van Cleaveng distributions of H / C and O / C, double bond equivalents and aromaticity indices, and main peak bandwidth / number of peaks. This fingerprint directly reflects molecular-level information such as the elemental composition, functional groups, and degree of condensation of Fe-OC.

[0012] First, water extraction is used to recover pore water or weakly adsorbed DOM or Fe-OC without altering the chemical environment, ensuring that this fraction represents only soluble / weakly bound states. Then, acid extraction (HCl) is used to remove carbonates and some proton-promoted acid-soluble bindings, while reducing the inorganic salt / metal background, creating a cleaner matrix for subsequent selective reduction. Finally, near-neutral reduction extraction with sodium bicarbonate-sodium dithionite is used to selectively dissolve the reduced soluble bound states supported by Fe(III) oxides.

[0013] If acid extraction or near-neutral reduction extraction with sodium bicarbonate-sodium dithionite is performed first, the surface charge of the particles and the mineral phase will be irreversibly changed (dissolving / reducing Fe(III) oxides and releasing metal ions), causing the components that should originally belong to "water extraction / acid dissolution / reduction soluble" to be mixed in, and losing the orthogonality of the mechanism of "binding form and fraction".

[0014] The process of first performing near-neutral reduction extraction with sodium bicarbonate and sodium dithionite followed by acid extraction results in the rapid decomposition of residual sodium dithionite in acid, releasing SO2 / sulfite ions. This poses both safety risks and can trigger secondary oxidation and reprecipitation of Fe(II) and secondary adsorption of DOM, leading to quantitative and spectroscopic deviations. Furthermore, the premature near-neutral reduction extraction with sodium bicarbonate and sodium dithionite introduces a large amount of reducing agent, sulfite (sulfite) ions, and buffer salts, significantly increasing metal quenching and internal filtration background in EEM and salt clusters and ion suppression in HRMS. This disrupts the low-salinity interface between subsequent SPE (solid phase extraction) and EEM and HRMS.

[0015] The extraction sequence employed in this invention follows the order of first the soluble state, then the acid-soluble state, and finally the reducible state, ensuring cross-laboratory comparability and reproducibility. Therefore, the three extraction steps in this invention cannot be interchanged.

[0016] Furthermore, in step S1, the sediment is air-dried sediment, and the mass ratio of the air-dried sediment to the volume of the water is 1g:20mL-1g:40mL.

[0017] Furthermore, in step S2, the concentration of the hydrochloric acid aqueous solution is 0.2 mol / L-1 mol / L, and the volume ratio of the added hydrochloric acid aqueous solution to the mass ratio of the air-dried sediment is 20 mL:1 g - 40 mL:1 g.

[0018] Furthermore, in step S3, the molar ratio of sodium bicarbonate to sodium dithionite in the aqueous solution is 1:1.

[0019] In step S3, adjusting the pH to 6.5-7.5 with a pH adjuster is to eliminate the interference of residual hydrochloric acid from step S2 on the originally neutral aqueous solution of sodium bicarbonate and sodium dithionite, thus affecting the reduction effect.

[0020] Furthermore, in the aqueous solution of sodium bicarbonate and sodium dithionite, the molar concentration of sodium bicarbonate or sodium dithionite is 0.05 mol / L to 0.2 mol / L; the volume ratio of the added aqueous solution of sodium bicarbonate and sodium dithionite to the mass ratio of the air-dried sediment is 20 mL:1 g to 40 mL:1 g.

[0021] Furthermore, in steps S3 and S4, the pH adjuster includes an aqueous solution of hydrochloric acid or an aqueous solution of sodium bicarbonate; the filtration uses a filter membrane with a pore size of 0.22µm.

[0022] Furthermore, in steps S1-S3, the stirring time is the same; the volumes of water, hydrochloric acid aqueous solution, sodium bicarbonate and sodium dithionite aqueous solution added are the same; and the rotation speed and time used for centrifugation are the same.

[0023] Standardizing the sample volume, extraction liquid volume, stirring, centrifugation, and pH control can improve the reproducibility and comparability of test structures across batches or laboratories.

[0024] Furthermore, the stirring time was 1 hour; the volume of water, hydrochloric acid solution, sodium bicarbonate and sodium dithionite solution added was 30 mL; the centrifugation speed was 7000 rpm and the centrifugation time was 5 min.

[0025] Furthermore, in step S5, the specific operations include: first activating the SPE column with methanol, then equilibrating it with acidified ultrapure water (pH≈2); subsequently loading the acidified mixture and thoroughly rinsing with acidified ultrapure water to remove salt and metal; then eluting the retained DOM with methanol to obtain DOM methanol eluent.

[0026] Furthermore, the SPE column includes a PPL solid-phase extraction column.

[0027] PPL (styrene-divinylbenzene polymer) packing material possesses a composite structure of nonpolar (benzene ring skeleton) and weakly polar (divinylbenzene), enabling it to bind to different types of DOM molecules through various mechanisms such as hydrophobic interactions, π-π interactions, and van der Waals forces. High-quality PPL packing material undergoes rigorous purification, resulting in minimal leaching of organic impurities. In subsequent high-sensitivity mass spectrometry analyses (such as FT-ICR MS), it minimizes background noise and interference signals, ensuring that the detected molecules are genuine DOM molecules, rather than contaminants from the column packing material.

[0028] In summary, the advantages and beneficial effects of the present invention are as follows: This invention provides a sample pretreatment method for constructing molecular fingerprints of iron-bound organic carbon (OCC) in sediments. The sediments are sequentially extracted through water extraction, acid extraction, and near-neutral reducing extraction, following a sequence from weak to strong binding forces and from non-destructive to selectively destructive processes to extract iron-bound organic carbon (OCC). This method not only distinguishes Fe-OC fractions controlled by different binding forces and mineral phases, but also allows for unified solid-phase extraction desalination of the extracted samples. Furthermore, it is compatible with both energy-based fluorescence spectroscopy (EEM) and high-resolution mass spectrometry (HRMS) for molecular fingerprint characterization, enabling data verification and molecular fingerprint acquisition using both EEM and HRMS on the same sample. Attached Figure Description

[0029] Figure 1 Figures showing the results of EEM testing on samples obtained in the examples and comparative examples; Figure 2 The results of HRMS testing on samples obtained in the examples and comparative examples are shown in the figure. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0031] Materials and consumables used in the embodiments of this invention: ultrapure water (18.2 MΩ·cm); hydrochloric acid solution (0.5 mol·L⁻¹). -1 PPL solid-phase extraction column (100 mg level or equivalent adsorption resin), acidified ultrapure water (pH about 2), HPLC grade methanol; 0.22 µm filter membrane; PTFE / PEEK centrifuge tubes, low adsorption pipette tips, etc.

[0032] Example S1: Weigh 1.00 g of air-dried sediment into a centrifuge tube, add 30 mL of ultrapure water (18.2 MΩ·cm, liquid-to-solid ratio approximately 30 mL·g). -1 The mixture was shaken on a shaker for 1 hour; then centrifuged at 7000 rpm for 5 minutes, and the supernatant A was collected. The solid sludge after centrifugation was the "water extraction residue", i.e., solid sludge A, which was transferred to the next step of acid extraction.

[0033] The purpose of step S1 is to recover pore water and soluble / weakly adsorbed Fe-OC / DOM under conditions that do not significantly alter the chemical environment, for characterizing the most easily migrating organic matter fractions; while retaining the solid phase for subsequent fractionation by stronger interactions.

[0034] S2: Using solid precipitate A as the starting material, add 30 mL of 0.5 mol·L⁻¹ solution to the centrifuge tube. -1 Hydrochloric acid (HCl) aqueous solution was used as the extraction reagent and the mixture was shaken on a shaker for 1 h; then it was centrifuged at 7000 rpm for 5 min to obtain supernatant B and solid sludge B.

[0035] The purpose of step S2 is to selectively release acid-soluble bound Fe-OC / DOM (such as desorption caused by proton substitution and bridging metal dissociation) and remove acid-sensitive phases such as carbonates, creating a cleaner matrix for the next near-neutral reduction step and avoiding cross-contamination of fractions from different mechanisms.

[0036] S3: Using solid sludge B as the starting material, add 30 mL of 0.11 mol·L⁻¹ solution. -1 An aqueous solution of bicarbonate and sodium dithionite (formulation: dissolve 9.58 g of Na2S2O4 (sodium dithionite) and 4.62 g of NaHCO3 (sodium bicarbonate) in 500 mL of solution); monitor the pH with a pH meter and adjust the pH to approximately 7 by micro-titration with dilute hydrochloric acid or sodium bicarbonate solution (preferably prepared fresh, protected from light, and under inert gas protection conditions); shake on a shaker for 1 h; then centrifuge at 7000 rpm for 5 min to obtain supernatant C and residue.

[0037] The purpose of step S3 is to selectively reduce and dissolve reducible phases such as Fe(III) (oxygen) hydride under near-neutral conditions, releasing the strongly adsorbed / coprecipitated organic matter (reducing soluble Fe-OC), while avoiding secondary chemical changes caused by strong acids and salt loading of spectrometry / mass spectrometry, and maintaining interface compatibility with subsequent EEM / MS.

[0038] S4: Filter supernatant A, supernatant B, and supernatant C separately using a microporous membrane with a pore size of 0.22 µm; take 5 mL of each for rapid detection of iron and dissolved organic carbon content. Mix the remaining supernatant A, supernatant B, and supernatant C thoroughly, and slowly add dilute hydrochloric acid (e.g., 1 mol·L⁻¹) dropwise using a pipette. -1 Add HCl and mix well to acidify the sample to a pH close to 2, thus obtaining an acidified mixture.

[0039] The purpose of step S4 is to adjust the sample to a uniform acidity to improve the retention of DOM (dissolved organic matter) on PPL and stabilize the metal complex morphology, laying the foundation for desalting / metal removal and batch-to-batch consistency.

[0040] S5: Install the PPL solid phase extraction column on a vacuum manifold / solid phase extraction device, activate it with methanol first, and then balance it with acidified ultrapure water (pH≈2); then load the acidified mixture obtained in step (4), and rinse thoroughly with acidified ultrapure water to remove salt / metal; then elute the retained DOM with 6-10 mL of methanol. If necessary, the methanol eluent can be filtered again at 0.22 µm to obtain the DOM methanol eluent.

[0041] The purpose of step S5 is to achieve thorough desalting / acid removal / metal background removal and DOM enrichment, providing a universal pretreatment method with low salt, low ionic intensity and comparable composition for EEM and high-resolution mass spectrometry.

[0042] S6: Place the DOM methanol eluent on a nitrogen blower and slowly blow it with nitrogen at a temperature of less than or equal to 40°C to remove most of the methanol and obtain a concentrated test sample with low residual alcohol.

[0043] The purpose of step S6 is to reduce the background and quenching effect of organic solvents, avoid the signal suppression / spectral peak interference of methanol on EEM and MS, and control the temperature to prevent thermal degradation.

[0044] Comparative Example (1) Weigh 1.00 g of air-dried sediment into a centrifuge tube, add 30 mL of ultrapure water (18.2 MΩ·cm, liquid-to-solid ratio approximately 30 mL·g). -1 The mixture was shaken on a shaker for 1 hour; then centrifuged at 7000 rpm for 5 minutes, and the supernatant A was collected. The solid sludge after centrifugation was the "water extraction residue", i.e., solid sludge A, which was transferred to the next step of acid extraction.

[0045] (2) Using solid sludge A as the starting material, add 30 mL of 0.5 mol·L⁻¹ solution to the centrifuge tube. -1Hydrochloric acid (HCl) aqueous solution was used as the extraction reagent and the mixture was shaken on a shaker for 1 h; then it was centrifuged at 7000 rpm for 5 min to obtain supernatant B and solid sludge B.

[0046] (3) Using solid sludge B as the starting material, add 30 mL of 0.11 mol·L⁻¹ solution. -1 An aqueous solution of bicarbonate and sodium dithionite (formulation: dissolve 9.58 g of Na2S2O4 (sodium dithionite) and 4.62 g of NaHCO3 (sodium bicarbonate) in 500 mL of solution); monitor the pH with a pH meter and adjust the pH to approximately 7 by micro-titration with dilute hydrochloric acid or sodium bicarbonate solution (preferably prepared fresh, protected from light, and under inert gas protection conditions); shake on a shaker for 1 h; then centrifuge at 7000 rpm for 5 min to obtain supernatant C and residue.

[0047] (4) Filter supernatant A, supernatant B and supernatant C separately using a microporous membrane with a pore size of 0.22 µm; take 5 mL of each for rapid detection of iron and dissolved organic carbon content, and mix the remaining supernatant A, supernatant B and supernatant C evenly, and use the mixture as the test sample.

[0048] EEM testing: The test samples obtained in the examples or comparative examples were transferred using a pipette and placed in a volumetric flask. The flask was then diluted to the target volume with ultrapure water to achieve a DOC (dissolved organic carbon) concentration of 2-5 mg C·L⁻¹. -1 A pH meter was used to titrate the sample with dilute hydrochloric acid or dilute sodium hydroxide solution to maintain the pH at 6-8, thus obtaining an aqueous sample for EEM testing.

[0049] Using a three-dimensional fluorescence spectrometer, the PARAFAC-compatible excitation / emission wavelength range and step size were set, and measurements were performed on aqueous samples. The volume and dilution factor were recorded, and internal filtration effect (IFE) correction and dilution linearity checks were performed during data processing to obtain an EEM dataset that can be used for parallel factor analysis.

[0050] HRMS testing: Test samples obtained from examples or comparative examples are directly injected, or the concentrated test sample is injected using a reconstituted solution at a mass ratio of 1:1 to the solvent (MeOH / H2O).

[0051] Statistical and scoring methods: EEM Indicator Definition and Scoring.

[0052] (1) S / N score (1-5): calculated as peak intensity / baseline noise (RMS); threshold suggestions: 1 (<5), 2 (5-10), 3 (10-20), 4 (20-50), 5 (>50).

[0053] (2) Severity of internal filtration effect (IFE) (high / medium / low): Based on the absorbance and the difference in intensity before and after correction, the A254 / Em cross absorbance >0.1 is recorded as "high", 0.05-0.1 is "medium" and <0.05 is "low".

[0054] (3) Metal quenching indicators (present / weak / absent): The relative intensity of the humic peak (such as C, A region) is used to determine the recovery after spiking / demetallization.

[0055] (4) PARAFAC fit score (1-5): can be calculated using Core Consistency or explained variance; threshold suggestions: 1 (<50%), 2 (50-70%), 3 (70-85%), 4 (85-95%), 5 (>95%).

[0056] Mass spectrometry index definition: Effective peak number: Number of deisotope / depolymer peaks with S / N>5; Salt cluster intensity percentage: Identify clusters with an interval of 22.99±0.2 Da and calculate their intensity as a percentage of the total ion current; RSD: Median relative standard deviation of peak intensity from three repeated injections.

[0057] EMM test results are as follows Figure 1 As shown, the HRMS test results are as follows: Figure 2 As shown, Figure 2 Part a of the diagram shows the HRMS test results obtained from the comparative analysis. Figure 2 Part b shows the HRMS test results obtained in the example.

[0058] Table 1 Qualitative and semi-quantitative results of HRMS testing

[0059] like Figure 1 And as shown in Table 1, the comparative examples ( Figure 1 In Part a), EEM measurements all exhibited significant metal quenching and internal filtering effects, manifested as suppressed peak shape, background enhancement, or drift. Example ( Figure 1 (Part b) Under uniform low ionic strength and pH conditions, the signal-to-noise ratio and peak shape stability of EEM are significantly improved, and the goodness of fit of parallel factor analysis (PARAFAC) is enhanced.

[0060] Table 2 Qualitative and semi-quantitative results of HRMS testing

[0061] like Figure 2 As shown in Table 2, the number of effective peaks and repeatability of the mass spectrometry in the examples were improved compared to the comparative examples. Figure 2 As shown, the test samples obtained in the comparative example exhibited typical salt clusters and ion suppression in high-resolution mass spectrometry; the test results obtained in the example showed a significant reduction in salt clusters and an increase in the number of effective peaks.

[0062] The above descriptions are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the above claims.

Claims

1. A sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments, characterized in that, Includes the following steps: S1: Mix the sediment with water and stir until all Fe-OC or DOM that can be leached by water are dissolved; centrifuge to obtain supernatant A and solid sediment A. S2: Mix the solid sludge A with hydrochloric acid aqueous solution, stir until the reaction is complete, centrifuge, and obtain supernatant B and solid sludge B; S3: Mix the solid sludge B with an aqueous solution of sodium bicarbonate and sodium dithionite, adjust the pH to 6.5-7.5 using a pH adjuster, stir until the reaction is complete, centrifuge, and obtain the supernatant C and residue; S4: Filter the supernatant A, supernatant B and supernatant C; mix them evenly, add a pH adjuster to adjust the pH of the mixture to 1.5-2.5 to obtain an acidified mixture; S5: The acidified mixture is subjected to solid-phase extraction for desalting and elution to obtain an eluent; S6: Concentrate the eluent with nitrogen to obtain a test sample; divide the test sample into two parts for fluorescence three-dimensional spectroscopy analysis and high-resolution mass spectrometry analysis, respectively.

2. The sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments according to claim 1, characterized in that, In step S1, the sediment is air-dried sediment, and the mass ratio of the air-dried sediment to the volume of the water is 1g:20mL-1g:40mL.

3. The sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments according to claim 2, characterized in that, In step S2, the concentration of the hydrochloric acid aqueous solution is 0.2 mol / L-1 mol / L, and the volume ratio of the added hydrochloric acid aqueous solution to the mass ratio of the air-dried sediment is 20 mL:1 g-40 mL:1 g.

4. The sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments according to claim 2, characterized in that, In step S3, the molar ratio of sodium bicarbonate to sodium dithionite in the aqueous solution is 1:

1.

5. The sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments according to claim 4, characterized in that, In the aqueous solution of sodium bicarbonate and sodium dithionite, the molar concentration of sodium bicarbonate or sodium dithionite is 0.05 mol / L to 0.2 mol / L; the volume ratio of the added aqueous solution of sodium bicarbonate and sodium dithionite to the mass ratio of the air-dried sediment is 20 mL:1 g to 40 mL:1 g.

6. The sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments according to claim 1, characterized in that, In steps S3 and S4, the pH adjuster includes an aqueous solution of hydrochloric acid or an aqueous solution of sodium bicarbonate; the filtration uses a filter membrane with a pore size of 0.22µm.

7. The sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments according to claim 1, characterized in that, In steps S1-S3, the stirring time is the same; the volumes of water, hydrochloric acid solution, sodium bicarbonate and sodium dithionite solution added are the same; and the rotation speed and time used for centrifugation are the same.

8. The sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments according to claim 7, characterized in that, The stirring time was 1 hour; the volume of water, hydrochloric acid solution, sodium bicarbonate and sodium dithionite solution added was 30 mL; the centrifugation speed was 7000 rpm and the centrifugation time was 5 min.

9. The sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments according to claim 1, characterized in that, In step S5, the specific operations include: first activating the SPE column with methanol, then equilibrating it with acidified ultrapure water; then loading the acidified mixture and thoroughly rinsing it with acidified ultrapure water to remove salt / metal; and then eluting the retained DOM with methanol to obtain DOM methanol eluent.

10. The sample pretreatment method for constructing iron-bound organic carbon molecular fingerprints in sediments according to claim 9, characterized in that, The SPE column includes a PPL solid-phase extraction column.

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