Method for determining nitrate content of pectin product

Abstract

The invention relates to the technical field of analytical chemistry detection, in particular to a method for determining the nitrate content of a pectin product, which comprises the following steps: step 1, breaking a micelle structure by gradient centrifugation, dissolving a pectin sample in ultrapure water to form a colloidal solution, and executing two-stage gradient centrifugation; step 2, performing low-temperature digestion with viscosity fed back in real time, adding a first digestion agent into the merged supernate, oscillating at the temperature lower than the initial temperature of thermal decomposition of pectin, and monitoring the viscosity of the solution in real time; step 3, biphase penetration suction filtration purification: mixing the digestion solution with a specific polar organic solvent, and standing to form a three-layer system; and step 4, interference window locking chromatography detection: adopting a high-capacity anion exchange chromatographic column, setting a gradient elution program based on retention time of pectin characteristic interference anions, dynamically adjusting suppressor current in a nitrate retention time window, and measuring nitrate peak area. Chromatographic detection is locked through an interference window, so that interference ions are eliminated, and the accuracy and reliability of nitrate radical determination are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry detection, in particular to a method for determining the nitrate content of a pectin product. Background Art

[0002] Pectin, a widely used gelling agent and stabilizer in the food industry, can be contaminated with nitrates during its raw material production. Because nitrate can be converted into carcinogenic nitrosamines in the human body, EU Regulation EC1881 / 2006 explicitly limits the amount of nitrate in food additives to ≤50mg / kg. Therefore, establishing an accurate method for determining the nitrate content of pectin products is crucial to ensuring food safety.

[0003] Currently, the detection of nitrate mainly includes colorimetry, ion chromatography and electrode method. However, these methods have the following insurmountable defects when applied to pectin matrix: Foaming and nitrate loss: The high viscosity of pectin leads to the formation of a stable gas-liquid colloidal system in the early stages of digestion. When using traditional boiling water digestion or high-temperature ashing, the sudden increase in local vapor pressure causes foaming.

[0004] Colloidal adsorption effect: Incompletely degraded pectin residues (molecular weight > 10 kDa) adsorb nitrate ions through hydrogen bonding and electrostatic interactions. Studies have shown that for every 1% increase in pectin residue, nitrate recovery decreases by 2.8-4.5 percentage points.

[0005] Turbid digestion solution blocks the flow path: The colloidal particles (particle size 0.2-5μm) remaining in the pectin digestion solution cannot be completely retained by conventional filter membranes. After entering the detection system, they block the flow path of the ion chromatography column, causing the column pressure to cumulatively increase until failure.

[0006] Viscosity hinders reaction mass transfer: The viscosity of pectin solutions (≥500 mPa·s) is significantly higher than that of conventional samples (<50 mPa·s), making it difficult for the digester to diffuse into the micelles. Experiments have shown that under a fixed temperature program, the diffusion rate of the digester in pectin colloids is only 18-22% of that in aqueous solutions.

[0007] Local overheating triggers thermal decomposition: The non-uniform thermal conductivity of pectin colloid causes a temperature gradient of >15°C in the digestion system. When the local temperature exceeds 130°C, nitrate ions undergo irreversible thermal decomposition (decomposition rate constant k = 0.047min -1 ), which directly causes the recovery rate to fluctuate by ±25%.

[0008] Therefore, there is an urgent need for a method for determining the nitrate content of pectin products to solve the above problems. Summary of the Invention

[0009] Based on the above purpose, the present invention provides a method for determining the nitrate content of a pectin product, comprising the following steps: step 1: gradient centrifugation to break the micelle structure, dissolving the pectin sample in ultrapure water to form a colloidal solution, and performing two-stage gradient centrifugation: in the first stage, operating under a centrifugal force for separating large-particle micelles to obtain a first supernatant; in the second stage, ultrapure water is added to the precipitate, and a second centrifugation is performed under a higher centrifugal force for separating small-particle micelles, and the two supernatants are combined; Step 2: Low-temperature digestion with real-time viscosity feedback: add the first digestant to the combined supernatant, shake at a temperature below the onset temperature of pectin thermal decomposition, and monitor the solution viscosity in real time; When the viscosity drops to the trigger threshold, the second digester is added and a step-by-step temperature increase program is started: the temperature is maintained constant after each temperature interval until the final digestion temperature is reached; Step 3: Two-phase penetration filtration purification: the digestion solution is mixed with a specific polar organic solvent and allowed to stand to form a three-layer system; The lower aqueous phase is extracted by penetrating means using a vacuum suction device, wherein the suction device is equipped with a filter membrane for retaining colloidal residues; Step 4: Interference window locked chromatography detection, using a high-capacity anion exchange column, setting a gradient elution program based on the retention time of the characteristic interfering anions of pectin, dynamically adjusting the suppressor current within the nitrate retention time window, and measuring the nitrate peak area.

[0010] Preferably, in step 1, the method for determining the centrifugal force of the two-stage gradient centrifugation comprises preparing a series of pectin concentration gradient solutions, centrifuging under increasing centrifugal force and measuring the transmittance of the supernatant; Draw a transmittance-centrifugal force change curve, and set the centrifugal force corresponding to the first inflection point of the curve as the centrifugal force of the first stage; Ultrapure water was added to the first-stage precipitation, and the dispersion was centrifuged a second time under increasing centrifugal force and the transmittance of the redispersed liquid was measured; The centrifugal force when the transmittance drops to a set percentage of the initial value is set as the second stage centrifugal force; The transmittance measurement is performed at a selected wavelength in the visible light region, and the average value of three consecutive measurement fluctuation values ​​less than a set threshold is used as the basis.

[0011] Preferably, in step 2, the design method of the step-by-step heating program includes: The weight loss rate curve of pectin was obtained by thermogravimetric analysis, and the temperature corresponding to the mutation point of the weight loss rate was identified; The temperature interval is divided based on the temperature of adjacent mutation points; Conduct constant temperature digestion experiments in various temperature ranges and draw viscosity versus time curves; The time required for the viscosity to drop to the set percentage of the maximum viscosity value in the temperature range is set as the constant temperature holding time of the range; The final digestion temperature is determined based on the fact that the fluctuation value of the light transmittance of the digestion solution measured three times in a row is less than a set threshold.

[0012] Preferably, in step 2, the method for determining the trigger threshold includes: The rotational viscometer torque values ​​were measured at fixed time intervals during the pre-digestion stage; Calculate the viscosity change rate of two adjacent measuring points and draw the change rate-time curve; When the slope of the curve approaches zero, the current viscosity value is taken as the trigger threshold; The rate of adding the second digester is as follows: when the viscosity is higher than the trigger threshold value, slow addition is adopted; when it is lower than the trigger threshold value, fast addition is adopted.

[0013] Preferably, in step 3, the method for selecting the specific polar organic solvent includes: Add the characteristic degradation product standard of pectin to ultrapure water, add the candidate solvent and shake to separate the layers; Measure the residual rate of target degradation products in the aqueous phase; Select a solvent whose residual rate is lower than a set threshold and whose refractive index difference with the water phase is greater than a set value; The pore size of the filter membrane of the vacuum suction device is determined in the following manner: a dynamic light scattering test is performed by eliminating the end point solution, and a set ratio of particle sizes at specific quantiles on the cumulative particle size distribution curve is used as a criterion for pore size selection.

[0014] Preferably, the penetrating extraction operation method includes providing a refractive index sensor at the bottom of the layered container to monitor the interface position in real time; When the sensor detects a sudden change in the refractive index, suction is initiated; The suction rate is dynamically adjusted according to the thickness of the interface layer: when the thickness is greater than the set value, low-speed suction is used, and when it is less than the set value, high-speed suction is used; The suction termination condition is that the sensor detects the characteristic signal of the refractive index of the organic phase.

[0015] Preferably, in step 4, the method for setting the gradient elution program includes analyzing the anion chromatogram of the blank pectin digestion solution to identify the pectin characteristic interference peak; Based on the retention time of nitrate, the half-peak width of the first interference peak is expanded forward by a set multiple, and the half-peak width of the second interference peak is expanded backward by a set multiple to form an elution program switching window; Elution was performed using a mobile phase with a high proportion of organic modifier within the window period, and elution was performed using a mobile phase with a high proportion of carbonate buffer outside the window period.

[0016] Preferably, the method for adjusting the suppressor current includes: When the elution program switching window is open, the background conductivity value is continuously measured; Calculate the first-order derivative of the background conductance over time; When the derivative value exceeds the set threshold, the suppressor current is adjusted according to the mapping relationship between the derivative value and the current increment; The mapping relationship is established through a preliminary experiment: increasing concentrations of pectin interfering substances are added to a nitrate standard solution, and corresponding data sets of background conductivity change rate and optimal detection current are recorded.

[0017] Preferably, the method further includes coordinated control of step 2 and step 3, specifically including: The digestion endpoint transmittance in step 2 is used as the input parameter for selecting the filter membrane pore size in step 3; When the transmittance is greater than the set value, a filter with a larger pore size is selected; when it is less than the set value, a filter with a smaller pore size is selected. The aperture adjustment range is linearly determined according to the degree to which the transmittance deviates from the set value.

[0018] Preferably, the method further includes associated control of step 1 and step 4, specifically including: The viscosity value of the combined supernatant in step 1 is used to correct the elution program switching window width in step 4; When the viscosity is higher than the set value, the window width is increased, and when it is lower, the window width is decreased; The width adjustment amount is calculated based on the difference between the viscosity value and the standard viscosity.

[0019] Beneficial effects of the present invention 1. The present invention avoids overheating caused by high temperatures through low-temperature digestion and real-time viscosity feedback. It can also accurately monitor viscosity changes. When the viscosity drops to a trigger threshold, the second digester is added. This measure effectively controls foam formation during the digestion process, reduces nitrate loss, and ensures the accuracy of the test results.

[0020] 2. The present invention breaks the micelle structure through gradient centrifugation and effectively separates large-size and small-size micelles using two-stage centrifugation, thereby removing incompletely degraded pectin residual chains to the greatest extent, thereby reducing the interference of the colloidal adsorption effect on nitrate ions and improving the recovery rate.

[0021] 3. This invention utilizes a two-phase penetrating filtration purification technology. By introducing a specific polar organic solvent and performing penetrating extraction using a vacuum suction device, colloidal particles in the digestion solution are effectively removed. The pore size of the filter membrane is dynamically selected based on the particle size distribution characteristics, ensuring that the flow path is not blocked, thereby ensuring the normal performance of chromatographic analysis.

[0022] 4. The present invention ensures that the digester effectively penetrates the pectin colloid by real-time monitoring of viscosity changes and combining it with low-temperature digestion. This technology ensures uniformity in the digestion reaction and reduces experimental errors caused by uneven reaction mass transfer.

[0023] 5. This invention combines a stepped temperature ramp with viscosity feedback control to precisely control temperature changes during the digestion process. The temperature ramp and temperature hold for each temperature interval are based on thermogravimetric analysis data and viscosity monitoring results, ensuring that the temperature gradient does not exceed 15°C. This prevents local overheating from affecting the thermal decomposition of nitrate, significantly improving the stability and accuracy of the assay results. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0025] Figure 1 is a flow chart of the steps of the method of the present invention; Figure 2 This is a flowchart of the steps of the method for determining the trigger threshold value according to the method of the present invention; Figure 3 This is a flowchart of the coordinated control of step 2 and step 3 of the method of the present invention. DETAILED DESCRIPTION

[0026] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. It is also noted that, to provide a more detailed description, the following embodiments are best and preferred embodiments, and those skilled in the art may employ alternative methods for implementing certain known technologies. Furthermore, the accompanying drawings are intended only to provide a more detailed description of the embodiments and are not intended to limit the present invention.

[0027] See Figure 1-Figure 3 An embodiment of the present invention provides a method for determining the nitrate content of a pectin product. In step 1, a pectin sample is first dissolved in ultrapure water to form a colloidal solution. Then, large-particle micelles and small-particle micelles are effectively separated by two-stage gradient centrifugation. In the first stage, large-particle micelles are separated at a lower centrifugal force to obtain a first supernatant; in the second stage, small-particle micelles are separated by increasing the centrifugal force to obtain a purer supernatant. This method avoids the presence of large particles in the pectin sample through staged and particle size-based centrifugation operations, reduces the interference of incompletely degraded pectin on nitrate determination, and ensures recovery and accuracy.

[0028] In step 2, after adding the first digester to the combined supernatant, the digestion process is carried out at a temperature below the starting temperature of pectin thermal decomposition, and the entire process is oscillated, and the viscosity of the solution is monitored in real time. Through real-time viscosity feedback, when the viscosity drops to a preset threshold, the second digester is added dropwise, and a step-by-step temperature increase program is initiated. The beneficial effect of this technical feature is that through real-time viscosity monitoring and control of the digestion temperature, nitrate decomposition caused by local overheating is avoided, the uniformity of the digestion reaction is ensured, and the error caused by uneven reaction mass transfer is effectively reduced.

[0029] In step 3, the digestate is mixed with a specific polar organic solvent to form a three-layer system, and a vacuum suction device is used to penetrate and extract the lower aqueous phase. This method effectively traps colloidal residues in the filter membrane, preventing colloidal particles in the digestate from entering the subsequent chromatographic analysis system and preventing flow path blockage. This method significantly improves the purity of the digestate, ensures the proper functioning of the ion chromatography column, and thus enhances the accuracy and stability of the analysis.

[0030] In step 4, a high-capacity anion exchange column is used in conjunction with a gradient elution procedure to dynamically adjust the suppressor current and measure the nitrate peak area within the nitrate retention time window. By setting a characteristic retention time for nitrate and precisely distinguishing and removing interfering anions in pectin, this step effectively mitigates the effects of interfering substances in pectin on nitrate analysis, improving the specificity and accuracy of the assay.

[0031] The combination of gradient centrifugation and low-temperature digestion minimizes the adsorption and loss of nitrate by pectin residues, ensuring stable recovery. Biphasic penetrating filtration purification technology successfully removes colloidal particles, preventing flow path blockage and column failure, ensuring a smooth analytical process. Interference window-locked chromatographic detection effectively eliminates interfering ions, improving the accuracy and reliability of nitrate determination.

[0032] In one possible embodiment, first, a series of pectin solutions with different concentrations are prepared, wherein the pectin concentrations of these solutions are sequentially increased, so that the pectin samples can exhibit different dispersion characteristics under different centrifugal forces.

[0033] The pectin solution was centrifuged under increasing centrifugal forces, and the transmittance of the supernatant was measured after each centrifugation. Changes in transmittance reflect the size, morphology, and dispersibility of the micelles. Transmittance measurements were performed using wavelengths in the visible light region to ensure accurate reflection of the micelles' optical properties.

[0034] The relationship between transmittance and centrifugal force is plotted on a curve. Transmittance typically increases before leveling off. The first inflection point of the curve, where the transmittance changes most significantly, corresponds to the centrifugal force of the first stage. This centrifugal force is optimal for separating larger micelles, effectively removing large particles and ensuring the accuracy of subsequent experiments.

[0035] Ultrapure water is added to the first-stage precipitate to form a diluted micelle solution, which is then centrifuged a second time. At this point, the centrifugal force continues to increase, and the small-sized micelles are separated from the precipitate by further centrifugation.

[0036] After the second centrifugation, measure the transmittance of the redispersed liquid. By observing the change in transmittance, determine the centrifugal force at which the transmittance drops to a set percentage of the initial value. This is the second-stage centrifugal force. This step ensures the effective separation of smaller micelles by accurately measuring the transmittance of the redispersed liquid.

[0037] All transmittance measurements must be performed at the set wavelength in the visible light region, and the average value of three consecutive measurements with fluctuation values ​​less than the set threshold must be used to ensure data stability and accuracy.

[0038] This method not only improves the separation efficiency of pectin samples but also accurately sets the centrifugal force, optimizes the pectin processing process, and provides a reliable sample basis for subsequent nitrate content determination. This method has strong operability and reproducibility, making it suitable for large-scale pectin sample analysis.

[0039] In one possible implementation, a pectin sample is heated using thermogravimetric analysis (TGA), and its weight loss during heating is recorded. TGA generates a weight loss rate curve, which can be analyzed to identify sudden changes in the weight loss rate (i.e., points where the curve undergoes a significant change). The temperature ranges corresponding to these sudden changes are typically associated with pectin decomposition, melting, or other thermal transitions, and serve as an important basis for determining the digestion temperature.

[0040] Based on the thermogravimetric analysis results, adjacent mutation point temperatures were selected as boundaries to define multiple temperature intervals. Each interval represents a different temperature range within which constant-temperature digestion experiments were conducted. By distinguishing between different temperature intervals, different heating strategies can be adopted for different stages of pectin thermal decomposition, improving the efficiency and accuracy of the digestion process.

[0041] In each temperature range, a constant-temperature digestion experiment was conducted, with the pectin solution continuously shaken and the viscosity curve plotted over time. Changes in viscosity reflect changes in the pectin's molecular structure, with a gradual decrease in viscosity typically indicating the progress of pectin decomposition and reactions. By monitoring viscosity changes in real time, the dynamics of the digestion process can be effectively monitored, ensuring that the reactions at each stage are fully progressing.

[0042] In each temperature range, the time required for the viscosity to drop to a set percentage of the maximum viscosity value in that range is set as the constant temperature hold time for that range. By precisely controlling the temperature range and constant temperature hold time, we can ensure that each stage of the digestion reaction proceeds under optimal conditions, avoiding over- or under-reaction and thus improving measurement accuracy.

[0043] The final digestion temperature is selected based on the transmittance fluctuation of the digestion solution. At the end of the digestion process, the transmittance fluctuation of the digestion solution is measured three times in a row and the average value is calculated. When the transmittance fluctuation value is less than the set threshold, the digestion process has stabilized, and the corresponding temperature is the final digestion temperature.

[0044] By combining thermogravimetric analysis with real-time viscosity monitoring, the heating conditions at each stage are precisely controlled, the pectin digestion process is optimized, and the efficient, accurate, and stable determination of the pectin nitrate content is ensured.

[0045] In one possible embodiment, during the pre-digestion stage, the torque of the pectin solution is measured at regular intervals using a rotational viscometer. A rotational viscometer reflects the rheological properties of a solution by sensing its viscosity. Because viscosity is closely related to the molecular structure of the solution, changes in solution viscosity can provide an important basis for determining the digestion progress and reaction extent.

[0046] By calculating the viscosity change rate between two adjacent measurement points (i.e., the ratio of the viscosity difference to the time difference), we can determine how the viscosity change rate changes over time. This rate of change is plotted against time to analyze the trend. The slope of the curve represents the rate of viscosity change and reflects the reaction rate of the pectin solution during the digestion process.

[0047] When the slope of the viscosity change rate-time curve approaches zero, it indicates that the solution's reaction rate has gradually stabilized and entered the equilibrium phase of the digestion process. The viscosity value at this point is the trigger threshold. During the digestion process, the rate of change of viscosity is closely related to the progress of the reaction. Therefore, determining the trigger threshold can accurately reflect whether the digestion reaction has reached the expected level.

[0048] The rate at which the second digestant is added is determined by the solution's current viscosity, depending on the trigger threshold setting. When the solution's viscosity is higher than the trigger threshold by a multiple, the second digestant is added slowly. This is because high viscosity indicates a denser aggregation of pectin in the solution, requiring a slower addition rate to avoid an overly rapid or uneven reaction. Conversely, when the viscosity is lower than the trigger threshold by a multiple, faster addition is used, which accelerates the reaction and improves digestion efficiency.

[0049] By monitoring the viscosity change rate in real time, the progress of the digestion reaction can be accurately determined, and the digester addition rate can be adjusted accordingly. This real-time feedback control method avoids the errors of traditional empirical operation, making the digestion process more refined, the reaction more uniform, and the results more reliable.

[0050] The digester addition rate is adjusted based on the trigger threshold. Rapid addition at the initial stage of the reaction can quickly activate the reaction, while slow addition during the higher viscosity stage helps avoid overly rapid or incomplete reactions, thereby ensuring reaction uniformity. This step-by-step control method can improve digestion efficiency and stability.

[0051] In one possible embodiment, before selecting a solvent, a standard sample of characteristic pectin degradation products must first be prepared. This standard sample is typically a stable product formed when pectin degrades under specific conditions and serves as a basis for measurement and comparison. This standard sample is added to ultrapure water, along with the candidate solvent. By oscillating and layering, the candidate solvent and the aqueous phase separate, forming two distinct phases. This step serves to test the solubility of different solvents.

[0052] After the solvent and aqueous phases are separated, the residual rate of the target degradation product in the aqueous phase is measured. These target degradation products are produced during pectin degradation, and their concentration is crucial for subsequent nitrate content determination. Measuring the residual amount in the aqueous phase provides insights into the solvent's selectivity and solubility. If a solvent effectively reduces the residual amount of the target degradation product in the aqueous phase, it indicates that the solvent has a good solubility for that specific degradation product.

[0053] Select a solvent with a residual rate below a set threshold and a refractive index difference with the aqueous phase greater than a set value. This set residual rate threshold ensures that the selected solvent effectively extracts the target degradation product from the aqueous phase, minimizing the impact of unwanted components in the aqueous phase on experimental results. Furthermore, a solvent with a larger refractive index difference helps enhance the separation between the solvent and the aqueous phase, thereby optimizing solvent distribution and recovery efficiency.

[0054] After selecting the solvent, the solution is filtered using a vacuum suction device. The selection of the filter membrane pore size is crucial to ensure effective separation of particles in the solution. To achieve this, a dynamic light scattering (DLS) test is performed on the solution after the endpoint solution is eliminated. Dynamic light scattering analyzes the particle size distribution by measuring the change in the intensity of scattered light from particles in the solution. The filter membrane pore size is selected based on the set ratio of particle sizes at specific quantiles on the cumulative particle size distribution curve. This ensures that the particle size passing through the filter membrane meets the experimental requirements, preventing excessive or insufficient particles from being filtered, thereby ensuring the accuracy of subsequent measurements.

[0055] By precisely selecting specific polar organic solvents and optimizing the pore size of the filter membrane, the accuracy, efficiency, and stability of the determination method for the nitrate content of pectin products can be effectively improved, thereby providing reliable data support for subsequent analysis.

[0056] In one possible embodiment, a refractive index sensor is installed at the bottom of the stratification container to monitor the position of the solution interface in real time. In a stratified solution, the refractive index of the aqueous and organic phases differs significantly, so the sensor can accurately detect changes in the interface position.

[0057] The refractive index sensor continuously monitors the position of the solution interface during the stratification process. Due to the significant difference in refractive index between the aqueous and organic phases in the solution, the sensor can identify changes in the position of the interface layer and respond promptly to sudden changes in refractive index.

[0058] When the sensor detects a sudden change in the refractive index, it indicates a significant interface change, possibly indicating separation of the aqueous and organic phases or interface movement. At this point, the system automatically initiates the aspiration process, extracting the specific solvent layer from the layering container. Using the sudden change in refractive index as the basis for initiating aspiration, the aspiration process is ensured at the appropriate time, avoiding ineffective or premature aspiration.

[0059] The pumping rate is dynamically adjusted based on the thickness of the interfacial layer. When the interfacial layer is thick, a slow pumping rate is used. This is because a thicker interfacial layer indicates a more stable boundary between the aqueous and organic phases, and slow pumping helps better separate the layers and avoid excessive mixing. Conversely, when the interfacial layer is thin, a high pumping rate is used, which improves separation efficiency, rapidly extracts the solution, and minimizes solvent loss.

[0060] The aspiration process is terminated when the refractive index sensor detects the characteristic refractive index signal of the organic phase, indicating that the aqueous phase has been completely extracted and the target components in the organic phase have been fully separated. At this point, the aspiration process stops, ensuring maximum retention of the target components in the organic phase and avoiding over-aspiration.

[0061] By combining real-time monitoring of the refractive index sensor with dynamic adjustment of the suction rate, the solvent separation process can be efficiently and accurately controlled, thereby optimizing the determination process of the nitrate content of the pectin product and ensuring the accuracy and reliability of the experimental results.

[0062] In one possible implementation, a blank pectin digest is first analyzed using anion chromatography to obtain an anion chromatogram. This step identifies and calibrates the interfering peaks in the chromatogram, particularly those of characteristic interfering substances that may be present in pectin (such as carbohydrates and pectin degradation products), providing a basis for subsequent elution program design. The purpose of identifying interfering peaks is to ensure that these interfering components will elute with nitrate, thereby affecting the accurate determination of nitrate content.

[0063] Using the retention time of nitrate as a benchmark, set the switching window of the elution program according to the characteristics of the interfering peaks in the chromatogram. The specific operations are as follows: The first interfering peak's half-peak width is expanded forward by a set multiple to determine the start of the elution window, while the second interfering peak's half-peak width is expanded backward by a set multiple to determine the end of the window. This interference peak-based half-peak width expansion method ensures that during chromatographic separation, the elution window accurately covers the peak region that may interfere with nitrate separation, thereby reducing the impact of these interfering peaks on the measurement results.

[0064] During the elution window, a high proportion of organic modifiers (such as acetonitrile, methanol, etc.) is used as the mobile phase for elution. This is because organic modifiers can effectively adjust the polarity of the solvent, enhance the separation of nitrates, and reduce the interference of interfering components.

[0065] Outside the window period, high-ratio carbonate buffer is used for elution. Carbonate buffer has a high buffering capacity, which can effectively maintain the stability of the chromatographic column and prevent interference from other irrelevant components.

[0066] Based on the elution window and mobile phase settings, gradient elution is performed. Within the window period, the proportion of organic modifier in the mobile phase gradually increases, helping to separate nitrate from interfering substances. Outside the window period, the carbonate buffer elutes other irrelevant substances, ensuring accurate nitrate determination.

[0067] The precisely designed gradient elution procedure effectively reduces the impact of interfering substances in pectin products on nitrate content determination, thereby improving the accuracy, precision, and efficiency of the determination. This method provides a reliable technical means for pectin product quality control.

[0068] In one possible implementation, while the elution program switching window is active, changes in sample conductivity during chromatographic analysis are monitored in real time by continuously measuring background conductivity. Background conductivity reflects the concentration and properties of ions in the sample, so any conductivity changes caused by pectin interference can be captured in real time through this process.

[0069] Based on the measured background conductivity, the first-order derivative of the background conductivity over time is calculated. This derivative reflects the rate of conductivity change and, in turn, reveals the dynamics of ionic composition in the sample. When the derivative value exceeds a set threshold, it indicates a significant conductivity change, likely due to interfering components (such as pectin), which could affect nitrate determination.

[0070] When the first-order derivative exceeds the set threshold, the suppressor current is adjusted based on a mapping between the derivative and the current increment. This mapping was obtained through preliminary experiments in which pectin interfering agents were gradually added to a nitrate standard solution. The effect of varying concentrations of the interfering agent on the rate of change in background conductivity was observed, and the corresponding data set between the optimal detection current and the rate of change in conductivity was recorded.

[0071] The preliminary experiment involves adding increasing concentrations of pectin interfering compounds to a nitrate standard solution and observing the change in background conductivity at different concentrations. The relationship between the rate of change in background conductivity and the optimal detection current is recorded, and this data set is used to establish a mapping between current and the rate of change in background conductivity. This mapping provides a basis for subsequent actual measurements, allowing precise adjustment of the suppressor current to accommodate varying interfering compound concentrations.

[0072] By dynamically adjusting the suppressor current, the impact of pectin interference on nitrate content determination can be effectively reduced, improving the accuracy and reliability of the results. This method optimizes every step of the determination process by monitoring changes in background conductivity in real time and combining it with mapping relationships established in preliminary experiments, offering significant technical advantages.

[0073] In one possible embodiment, when measuring the nitrate content of a pectin product, a digestion process is first performed to decompose impurities or interfering substances in the pectin product through heating, chemical reagents, or other means. The endpoint transmittance of the digestion process is used to determine whether the digestion is complete. The transmittance value reflects the content and degree of solubility of particulate matter in the solution. During the digestion process, changes in transmittance are monitored until the set endpoint transmittance is reached.

[0074] After the transmittance measurement is completed, the appropriate filter membrane pore size is selected based on the transmittance value. Depending on the transmittance, filters of different pore sizes are selected for further solution processing. When the transmittance is greater than the set value, it indicates that the solution contains few impurities, and a filter membrane with a larger pore size can be selected for filtration. Conversely, when the transmittance is less than the set value, it indicates that the solution may contain a large amount of particulate matter or dissolved matter, and a filter membrane with a smaller pore size should be selected for filtration to ensure that further operations are not interfered with by impurities.

[0075] The pore size selection is linearly adjusted based on the degree to which transmittance deviates from the set value. For example, if the transmittance deviates significantly from the set value, a wider pore size range can be selected, significantly optimizing the filtration effect. Specifically, the greater the deviation, the more precise the pore size difference needs to be. This allows for more precise removal of interfering substances from the solution, ensuring the accuracy of subsequent measurements.

[0076] By combining the digestion endpoint transmittance with the filter membrane pore size selection, the filtration process is optimized using linear adjustment, significantly improving the accuracy and reliability of nitrate content determination. This technical feature not only improves operational efficiency but also enhances the adaptability of the method and the accuracy of the data, and has important practical value.

[0077] In one possible embodiment, the combined supernatants from step 1 are first processed. During the digestion and separation process, the combined supernatants are affected by different components, causing their viscosity to change. Therefore, after combining the supernatants, their viscosity must first be measured. This viscosity value serves as the basis for adjusting the elution program switching window width in subsequent steps.

[0078] During the elution process in step 4, the width of the elution program's switching window significantly impacts the results. By properly controlling the switching window width, elution can be optimized, the retention of interfering substances in the sample can be reduced, and accurate nitrate determination can be ensured. In step 1, the viscosity of the supernatant is incorporated as an input parameter to modify the width of the elution program's switching window.

[0079] If the viscosity of the combined supernatant is higher than the set standard, it indicates that the solution is thick, which may affect the elution process of some components in the sample. Therefore, the width of the elution program switching window should be increased to better separate the components in the sample. If the viscosity of the combined supernatant is lower than the standard, it indicates that the solution is thin, and the window width should be narrowed to prevent excessive sample loss and ensure the accuracy of the measurement process.

[0080] The window width adjustment is calculated based on the difference between the viscosity value and the reference viscosity. For example, if the viscosity value is higher than the reference viscosity, the window width can be increased linearly by the difference. If the viscosity value is lower than the reference viscosity, the window width can be decreased linearly by the same amount. This proportional calculation ensures that each adjustment is tailored to the actual sample conditions, thereby optimizing the elution process.

[0081] By precisely adjusting the width of the elution program switching window, the determination process of nitrate content was optimized, which significantly improved the separation efficiency and determination accuracy. At the same time, human errors were reduced, the adaptability and operational efficiency of the method were enhanced, and it has high application value.

[0082] The embodiments of the present invention are primarily used for determining nitrate content in pectin products. Nitrate, as an important additive in pectin production, is subject to a certain degree of contamination during its preparation, so its content needs to be strictly controlled. The present invention provides a method for accurately determining nitrate content by adjusting the pore size of a filter membrane and combining viscosity and transmittance values. This method improves measurement accuracy and reduces operational complexity, making it suitable for quality monitoring of pectin in large-scale production.

[0083] This example specifically describes the determination process of nitrate content in pectin samples. The main equipment used in the experiment includes: Viscometer (Model: DV3T, Brookfield, Measuring range: 0.3-6,000,000 cP, Accuracy: ±1%) Light transmittance meter (model: UV-2600, SHIMADZU, measurement wavelength: 400nm-800nm) Filter membrane (pore size: 0.45 μm) Pectin sample: commercially available standard pectin containing a certain amount of nitrate was selected.

[0084] Nitrate standard solution: Use 10 mg / L nitrate standard solution.

[0085] Solvent: distilled water.

[0086] Experimental temperature: 25°C (±2°C).

[0087] Experimental time: The time of each step is strictly controlled within the specified range.

[0088] Specifically, 10 g of pectin sample was taken, 90 mL of distilled water was added, stirred evenly, and allowed to stand for 20 minutes to completely dissolve the pectin.

[0089] The viscosity of the sample solution was measured using a viscometer. According to preliminary experiments, when the viscosity of the pectin solution was between 0.5 cP and 1000 cP, the correlation between the transmittance measurement result and the nitrate content was the best.

[0090] Use a transmittance meter to measure the transmittance of the sample, set the measurement wavelength to 600nm, and record the transmittance data of each sample.

[0091] Dynamically adjust the pore size of the filter membrane based on the viscosity measurement value in step 2. The specific adjustment rules are as follows: When the viscosity value is less than 1 cP, use a filter membrane with a pore size of 0.45 μm.

[0092] When the viscosity value is between 1 cP and 10 cP, use a filter membrane with a pore size of 0.6 μm.

[0093] When the viscosity value is greater than 10 cP, use a filter membrane with a pore size of 0.8 μm.

[0094] The formula is as follows: Aperture adjustment = 0.45 + 0.15 × log (viscosity value); The viscosity value is the actual measured value of the sample solution (unit: cP).

[0095] The nitrate content of each sample was determined by linear regression of transmittance and viscosity. The regression equation is: Nitrate content = 0.005 × transmittance − 0.02 × viscosity + 0.15; The transmittance range is 0-100%, and the viscosity value unit is cP.

[0096] The experiment used five groups of pectin samples with different viscosities, tested their transmittance and nitrate content, and compared them with the traditional method (using only filter membranes without viscosity control). The experimental data table is as follows:

[0097] As can be seen from the table, the nitrate content measured by the method of the present invention is more accurate than that by the traditional method, with the error controlled within 5%. In particular, in high-viscosity samples, the traditional method leads to large measurement errors due to inappropriate selection of filter membrane pore size.

[0098] This embodiment significantly improves the accuracy of nitrate content determination by dynamically adjusting the filter membrane pore size and combining it with real-time viscosity and transmittance measurements. Conventional methods often use a one-size-fits-all approach to filter membrane pore size, failing to adapt to the needs of samples of varying viscosities. However, the present invention optimizes the filtration efficiency of samples of varying viscosities by matching the filter membrane pore size with the viscosity, thereby improving the accuracy of the measurement results.

[0099] Furthermore, experimental results show that the overall measurement error was reduced by approximately 30% using the method of the present invention, with the reduction being particularly significant in high-viscosity samples. This demonstrates that the method has greater adaptability and reliability when processing high-viscosity substances such as pectin products.

[0100] This example successfully optimizes the filter membrane pore size by combining dynamic adjustments of viscosity and transmittance, thereby improving the accuracy and reliability of the measurement. Compared with traditional methods, the experimental results show higher accuracy and smaller errors, which is a significant technical advantage, especially in applications with high-viscosity samples.

[0101] The embodiments of the present invention not only provide an accurate method for determining nitrate content, but can also be widely applied to other liquid sample analyses requiring filter membrane adjustment and viscosity control, and have high practical value and promotion potential.

[0102] The present invention encompasses any alternatives, modifications, equivalents, and solutions that fall within the spirit and scope of the present invention. To provide a thorough understanding of the present invention, specific details are described in detail in the following preferred embodiments of the present invention, but those skilled in the art will be able to fully understand the present invention without these detailed descriptions. Furthermore, to avoid unnecessary confusion regarding the essence of the present invention, well-known methods, processes, procedures, components, and circuits have not been described in detail.

[0103] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for determining the nitrate content of a pectin product, characterized in that: The following steps are involved: Step 1: Gradient centrifugation breaks down the micelle structure. The pectin sample is dissolved in ultrapure water to form a colloidal solution. A two-stage gradient centrifugation is performed: the first stage is operated under a centrifugal force that separates large-size micelles to obtain the first supernatant; In the second stage, ultrapure water is added to the precipitate, and the precipitate is centrifuged again at a higher centrifugal force to separate small-sized micelles, and the two supernatants are combined; Step 2: Low-temperature digestion with real-time viscosity feedback: add the first digestant to the combined supernatant, shake at a temperature below the onset temperature of pectin thermal decomposition, and monitor the solution viscosity in real time; When the viscosity drops to the trigger threshold, the second digester is added and a step-by-step temperature increase program is started: the temperature is maintained constant after each temperature interval until the final digestion temperature is reached; Step 3: Two-phase penetration filtration purification: the digestion solution is mixed with a specific polar organic solvent and allowed to stand to form a three-layer system; The lower aqueous phase is extracted by penetrating means using a vacuum suction device, wherein the suction device is equipped with a filter membrane for retaining colloidal residues; Step 4: Interference window locked chromatography detection, using a high-capacity anion exchange column, setting a gradient elution program based on the retention time of the characteristic interfering anions of pectin, dynamically adjusting the suppressor current within the nitrate retention time window, and measuring the nitrate peak area.

2. The method for determining the nitrate content of a pectin product according to claim 1, wherein: In step 1, the method for determining the centrifugal force of the two-stage gradient centrifugation comprises preparing a series of pectin concentration gradient solutions, centrifuging under increasing centrifugal force and measuring the transmittance of the supernatant; Draw a transmittance-centrifugal force change curve, and set the centrifugal force corresponding to the first inflection point of the curve as the centrifugal force of the first stage; Ultrapure water was added to the first-stage precipitation, and the dispersion was centrifuged a second time under increasing centrifugal force and the transmittance of the redispersed liquid was measured; The centrifugal force when the transmittance drops to a set percentage of the initial value is set as the second stage centrifugal force; The transmittance measurement is performed at a selected wavelength in the visible light region, and the average value of three consecutive measurement fluctuation values ​​less than a set threshold is used as the basis.

3. The method for determining the nitrate content of a pectin product according to claim 1, wherein: In step 2, the design method of the step-by-step heating program includes: The weight loss rate curve of pectin was obtained by thermogravimetric analysis, and the temperature corresponding to the mutation point of the weight loss rate was identified; The temperature interval is divided based on the temperature of adjacent mutation points; Conduct constant temperature digestion experiments in various temperature ranges and draw viscosity versus time curves; The time required for the viscosity to drop to the set percentage of the maximum viscosity value in the temperature range is set as the constant temperature holding time of the range; The final digestion temperature is determined based on the fact that the fluctuation value of the light transmittance of the digestion solution measured three times in a row is less than a set threshold.

4. The method for determining the nitrate content of a pectin product according to claim 1, wherein: In step 2, the method for determining the trigger threshold includes: The rotational viscometer torque values ​​were measured at fixed time intervals during the pre-digestion stage; Calculate the viscosity change rate of two adjacent measuring points and draw the change rate-time curve; When the slope of the curve approaches zero, the current viscosity value is taken as the trigger threshold; The rate of adding the second digester is as follows: when the viscosity is higher than the trigger threshold value, slow addition is adopted; when it is lower than the trigger threshold value, fast addition is adopted.

5. The method for determining the nitrate content of a pectin product according to claim 1, wherein: In step 3, the method for selecting the specific polar organic solvent includes: Add the characteristic degradation product standard of pectin to ultrapure water, add the candidate solvent and shake to separate the layers; Measure the residual rate of target degradation products in the aqueous phase; Select a solvent whose residual rate is lower than a set threshold and whose refractive index difference with the water phase is greater than a set value; The pore size of the filter membrane of the vacuum suction device is determined in the following manner: a dynamic light scattering test is performed by eliminating the end point solution, and a set ratio of particle sizes at specific quantiles on the cumulative particle size distribution curve is used as a criterion for pore size selection.

6. The method for determining the nitrate content of a pectin product according to claim 5, wherein: The penetrating extraction operation method includes providing a refractive index sensor at the bottom of the layered container to monitor the interface position in real time; When the sensor detects a sudden change in the refractive index, suction is initiated; The suction rate is dynamically adjusted according to the thickness of the interface layer: when the thickness is greater than the set value, low-speed suction is used, and when it is less than the set value, high-speed suction is used; The suction termination condition is that the sensor detects the characteristic signal of the refractive index of the organic phase.

7. The method for determining the nitrate content of a pectin product according to claim 1, wherein: In step 4, the method for setting the gradient elution program includes analyzing the anion chromatogram of the blank pectin digestion solution to identify the pectin characteristic interference peak; Based on the retention time of nitrate, the half-peak width of the first interference peak is expanded forward by a set multiple, and the half-peak width of the second interference peak is expanded backward by a set multiple to form an elution program switching window; Elution was performed using a mobile phase with a high proportion of organic modifier within the window period, and elution was performed using a mobile phase with a high proportion of carbonate buffer outside the window period.

8. The method for determining the nitrate content of a pectin product according to claim 7, wherein: The method for adjusting the suppressor current includes: When the elution program switching window is open, the background conductivity value is continuously measured; Calculate the first-order derivative of the background conductance over time; When the derivative value exceeds the set threshold, the suppressor current is adjusted according to the mapping relationship between the derivative value and the current increment; The mapping relationship is established through a preliminary experiment: increasing concentrations of pectin interfering substances are added to a nitrate standard solution, and corresponding data sets of background conductivity change rate and optimal detection current are recorded.

9. The method for determining the nitrate content of a pectin product according to claim 1, wherein: It also includes the coordinated control of step 2 and step 3, specifically including: The digestion endpoint transmittance in step 2 is used as the input parameter for selecting the filter membrane pore size in step 3; When the transmittance is greater than the set value, a filter with a larger pore size is selected; when it is less than the set value, a filter with a smaller pore size is selected. The aperture adjustment range is linearly determined according to the degree to which the transmittance deviates from the set value.

10. The method for determining the nitrate content of a pectin product according to claim 1, wherein: It also includes the associated control of step 1 and step 4, specifically including: The viscosity value of the combined supernatant in step 1 is used to correct the elution program switching window width in step 4; When the viscosity is higher than the set value, the window width is increased, and when it is lower, the window width is decreased; The width adjustment amount is calculated based on the difference between the viscosity value and the standard viscosity.

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