Flow injection dynamic chelation determination system and method for cyanide in wine and application

Abstract

The invention discloses a flow injection dynamic chelation determination system and method for cyanide in wine and application. The flow injection dynamic chelation determination system and method are used for eliminating interference of copper ions on cyanide detection on line. The system comprises a flow injection analyzer which is internally provided with a sample flow path, a reagent flow path and a peristaltic pump; the chelating agent flow path is coaxially converged with the sample flow path through a first three-way joint, so that the chelating agent is complexed with copper ions in a wine sample in real time, and cyanide is released; and the mixed solution is converged with the distillation reagent through the second three-way joint, is subjected to two-stage heating through the temperature control unit, and is subjected to colorimetric determination through the detection unit. The system adopts a closed inert polymer pipeline, the volume flow ratio of a wine sample to a chelating agent solution is (4: 1)-(5: 1), the anti-interference upper limit of copper ions is 2mg / L, the detection limit is 2mu g / L, and the recovery rate is 90%-110%. The measuring system and the measuring method are suitable for complex matrixes such as white spirit, yellow wine, grape wine and health care wine, additional hardware transformation is not needed, the cost is low, and the precision is high.

Description

Technical Field

[0001] The invention relates to the technical field of detection and analysis, and specifically proposes a flow injection dynamic chelation determination system, method and application of cyanide in wine. Background Art

[0002] Cyanide is one of the key toxic substances in the safety control of alcoholic beverages. Its accurate quantitative detection is crucial for controlling the quality and safety of alcoholic beverages and protecting the health of consumers. Currently, flow injection analysis (iFIA) is listed as one of the standard detection methods for cyanide in alcoholic beverages in the national standard (GB5009.36-2023 Method 5) due to its high efficiency and high degree of automation. However, in actual detection, the interference of the complexity of the wine sample matrix is ​​easily overlooked - especially the copper ions (Cu 2+ ) (Health wine, white wine, grape wine, yellow wine and other wines are brought in due to their raw materials, production process and storage process), will react with cyanide (CN - ) form a complex, which may prevent cyanide from being released by distillation or hinder the color reaction, resulting in a significantly low test result. This experimental study shows that copper ion standard solution is added to 1.0 mg / L in freshly distilled Luzhou-flavor liquor (cyanide content is 406.57 μg / L, copper is not detected), mixed and allowed to stand for 4 hours, and the cyanide content is tested according to the fifth method of GB5009.36-2023. The test result is only 69.15 μg / L.

[0003] The problem of copper ion interference in the conventional flow injection analysis of cyanide in wine is extremely hidden, specifically manifested in: ① The interference mechanism is complex: the complex equilibrium between copper ions and cyanide is affected by multiple factors such as the pH value and concentration of the wine sample, and it is difficult to completely eliminate it by conventional dilution. In addition, excessive dilution will lead to large fluctuations in cyanide detection results; ② Risk of false negative results: distorted cyanide detection results can easily be misjudged as "low cyanide content" wine samples, covering up actual safety hazards; ③ Method limitations: neither the current national standard method nor the methods reported in the literature provide an effective solution to the interference of copper ions in the cyanide flow injection analysis method.

[0004] Therefore, there is an urgent need for a measurement system and method with a simple structure, strong compatibility, and the ability to eliminate copper ion interference in real time. Summary of the Invention

[0005] In view of this, the present invention proposes a compact and highly adaptable flow injection dynamic chelation determination system for cyanide in wine. Without changing the national standard pretreatment process, the "dynamic online chelation" strategy is used to eliminate copper ion interference in real time, ensuring the accuracy and reliability of cyanide content determination.

[0006] This experimental study found that the binding ability of cyanide ions in wine samples to copper ions is weaker than that of ethylenediaminetetraacetic acid to copper ions. Based on this principle, the present invention effectively solves the problem of copper ion interference in the flow injection analysis of cyanide in wine by innovatively designing a "dynamic online chelation" flow system. The main advantages of the present invention are: ① Based on the traditional flow injection analysis, the disodium ethylenediaminetetraacetic acid mobile phase is introduced for the first time, so that the copper ions in the wine sample to be tested can be dynamically chelated online in the closed flow injection system, and the mixing ratio of the wine sample to be tested and the disodium ethylenediaminetetraacetic acid solution can be accurately controlled, effectively eliminating the interference of copper ions on the detection of cyanide in the wine sample to be tested. ② The present invention adopts flow pipeline design and flow path improvement optimization, without the need for major hardware modification, and has a high adaptability to the fully automatic flow injection analyzer, and does not affect the compatibility of the instrument control software.

[0007] The technical solution of the present invention is achieved as follows: The present invention provides a flow injection dynamic chelation determination system for cyanide in wine, comprising: Flow injection analyzer: Integrates sample flow path, reagent flow path and high-precision peristaltic pump to provide stable and continuous liquid drive.

[0008] The chelating agent flow path, which connects coaxially with the sample flow path outlet through the first T-joint, is used to inject a degassed disodium ethylenediaminetetraacetic acid (EDTA-2Na) solution into the wine sample. This chelating agent has a much higher complexing capacity with copper ions than cyanide ions, instantly displacing and releasing bound cyanide.

[0009] Flow ratio control module: Real-time adjustment of the volume flow ratio of wine sample to chelating agent solution to 4:1–5:1 to ensure maximum chelation efficiency while avoiding over-dilution.

[0010] Second three-way connector: The wine sample-chelating agent mixture and the distillation reagent are coaxially combined again to form a uniform reaction system.

[0011] Temperature control unit: equipped with a primary heating module (120°C) and a secondary heating module (85°C) to precisely control the distillation and color development reaction temperatures to ensure a sufficient and stable reaction.

[0012] Closed flow path design: All flow paths and T-joints are constructed from inert polymer tubing, such as FEP capillaries or Tygon tubing, with strictly matched inner diameters (0.6–1.3 mm for sample flow paths, 0.6–1.3 mm for chelating agents, and 0.5–1.5 mm for reagents), creating a closed system with no volatility or residue.

[0013] Peristaltic pump speed setting value: controlled at 15–30 rpm, taking into account both response time and detection sensitivity.

[0014] The present invention also provides a determination method comprising the following steps: S1 wine sample injection: The wine sample enters the sample flow path directly after national standard pre-treatment, without the need for additional offline steps.

[0015] S2 dynamic chelation: Degassed EDTA-2Na solution (concentration 0.1–1.0 g / L) is coaxially combined with the wine sample in the first three-way connector with a mixing time of ≤5 s. The copper ions are complexed in real time and the cyanide is completely released.

[0016] S3 distillation-color development: After the mixed liquid and the distillation reagent are combined at the second T-joint, they undergo a first-stage distillation at 120°C and a second-stage color development at 85°C to ensure efficient conversion of cyanide to hydrogen cyanide.

[0017] S4 colorimetric determination: The classic isonicotinic acid-barbituric acid method is used to perform colorimetric determination at a wavelength of 600 nm. The entire process is automatically recorded and the results are output by the flow injection optical system.

[0018] In some embodiments, the technical performance of the above-mentioned determination method is as follows: Anti-interference upper limit: When the copper ion concentration is ≤2mg / L, the deviation of cyanide determination results is <5%.

[0019] Detection limit: ≤2μg / L, significantly better than the current standard.

[0020] Precision: relative standard deviation (RSD) ≤ 2.5%.

[0021] Recovery rate: 90%–110%, covering complex matrices such as white wine, yellow wine, grape wine, and health wine.

[0022] Detection throughput: 15 samples / hour, meeting batch detection needs.

[0023] The present invention has the following beneficial effects compared to the prior art: Compared with existing technologies, the present method is the first to introduce a "dynamic online chelation" system for flow injection analysis of cyanide in alcoholic beverages. By introducing a disodium ethylenediaminetetraacetic acid chelating solution into the mobile phase, it achieves real-time chelation of copper ions in a closed flow path (avoiding cyanide loss from pre-chelation in an open system). Second, the innovative pipeline design ensures thorough dynamic mixing of the chelating agent and the alcohol sample while precisely controlling the mixing ratio. Third, the flow path is highly adaptable and can be achieved simply by adjusting the flow line specifications and improving the flow path. Fourth, the instrument parameter optimization system significantly improves the signal-to-noise ratio, bringing the method's upper limit for copper ion interference to 2 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 1 This is a schematic diagram of the link relationship of the flow injection dynamic chelation determination system for cyanide in wine of the present invention; Figure 2 It is a flow path diagram of a cyanide flow injection instrument in the prior art; Figure 3 This is a flow diagram of the flow injection dynamic chelation determination system for cyanide in wine of the present invention.

[0026] In the figure: 1-flow injection analyzer, 2-chelating agent flow path, 3-first three-way structure, 4-second three-way structure, 5-detection unit, 6-temperature control unit, 11-sample flow path, 12-reagent flow path, 13-peristaltic pump, 61-primary heating module, 62-secondary heating module. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] It should be noted that when an element is referred to as being “fixed on” or “disposed on” another element, it may be directly on the other element or indirectly on the other element. When an element is referred to as being “connected to” another element, it may be directly connected to the other element or indirectly connected to the other element.

[0029] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on this application.

[0030] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0032] Unless otherwise specified, the methods used in the following examples are conventional methods. The materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, unless otherwise specified, and can be obtained commercially by those skilled in the art.

[0033] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value, regardless of whether the range is disclosed alone. For example, when a range "1 to 5" is disclosed, the described range should be interpreted as including the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within the range. In the present specification and claims, range definitions may be combined and / or interchanged, and if not otherwise stated, such ranges include all subranges contained therein.

[0034] The flow injection instrument in the embodiment is a Beijing Jitian fully automatic flow injection analyzer (model FF-QHWL08).

[0035] Example 1 Determination of cyanide in Luzhou-flavor liquor.

[0036] Instrument and system construction A flow injection analyzer (1) was used, which had the following internal configuration: Sample flow path (11): FEP capillary with an inner diameter of 0.80 mm and a length of 110 cm, and Tygon pump tubing with an inner diameter of 1.30 mm and a length of 38 cm; Reagent flow path (12): distillation reagent segment FEP capillary with inner diameter of 0.60 mm and length of 110 cm, Tygon pump tube with inner diameter of 0.76 mm and length of 38 cm; Peristaltic pump (13): set value 22 rpm (instrument panel reading); Chelating agent flow path (2): FEP capillary with an inner diameter of 0.60 mm and a length of 110 cm, Tygon pump tube with an inner diameter of 0.76 mm and a length of 38 cm, the outlet end of which is connected to the inlet end of the first three-way connector (3); Second three-way connector (4): the outlet end of the first three-way connector (3) is coaxially connected to the outlet end of the reagent flow path (12); Temperature control unit (6): first-level heating module 120°C, second-level heating module 85°C; Detection unit (5): Flow injection optical system, wavelength 600nm.

[0037] All tubing and T-connectors are made of inert polymer (FEP / Tygon) to form a closed flow path.

[0038] ① Reagent preparation 0.5g / EDTA-2Na chelating solution: Weigh 0.5g EDTA-2Na, dissolve it in deionized water and dilute to 1000mL, prepare it before use, and degas it by ultrasonication for 15min.

[0039] Sodium hydroxide solution: 1.0 g / L, prepared according to GB5009.36-2023 31.2.1.

[0040] The distillation reagent, buffer solution, chloramine T solution and color developer were prepared according to GB5009.36-2023 31.2.1.

[0041] Cyanide standard solution: prepared according to GB5009.36-2023 31.4.

[0042] ②Pipeline assembly Sample tube: FEP capillary input tube with inner diameter of 0.80 mm × 110 cm, output tube with inner diameter of 0.80 mm × 22 cm, intermediate Tygon pump tube with inner diameter of 1.30 mm × 38 cm, connected in series with adapters.

[0043] Chelating agent solution mobile phase tube: FEP capillary input tube with inner diameter of 0.60 mm × 110 cm, output tube with inner diameter of 0.60 mm × 52 cm, middle Tygon pump tube with inner diameter of 0.76 mm × 38 cm, connected in series with adapters.

[0044] The rest of the pipelines are consistent with the original configuration.

[0045] ③Flow path improvement The sample output tube and the EDTA-2Na output tube are mixed via the first three-way connector (3).

[0046] The mixed liquid is then mixed with the distilled reagent via the second three-way connector (4) for subsequent analysis.

[0047] ④Parameter settings Peristaltic pump speed setting value: 22rpm Time to valve: 300s Injection time: 85s Sample cycle: 225s Injection time: 140s Cleaning time: 40s Injection needle cleaning time: 15s Primary heating module: 120°C Secondary heating module: 85℃ Front-end acquisition gain: 110K ⑤Sample selection and processing Take a batch of Luzhou-flavor liquor (copper ions were not detected). According to GB5009.36-2023 33.1.2: Accurately pipette 1.00 mL of sample into a 25 mL volumetric flask, dilute to the mark with 1.0 g / L sodium hydroxide solution, mix thoroughly, and let stand for 10 minutes to obtain the sample to be tested.

[0048] ⑥Testing of samples to be tested Scheme A: The measurement was performed using the improved flow path and optimized parameters (peristaltic pump setting 20 rpm, injection time 80 s, cleaning time 30 s, primary heating 118°C, front-end acquisition gain 100K).

[0049] Solution B: Use the improved flow path and optimized parameters (see ④) for measurement.

[0050] ⑦Results The same Luzhou-flavor liquor: Measured value by scheme A: 302.13 μg / L Solution B measurement value: 310.76 μg / L The deviation between the two methods was only 2.82%, indicating that the improved flow path and parameters can be directly applied and are fully compatible with the instrument control software.

[0051] Example 2 2.1 Sample preparation ① Prepare 100 mL of 50% vol ethanol aqueous solution (copper ions not detected).

[0052] ② Take part of the solution and place it in a 100mL volumetric flask, add 1mL of 50mg / L cyanide standard solution, dilute to 100mL with ethanol aqueous solution, and mix well to obtain sample 1 with a cyanide concentration of 500μg / L.

[0053] ③ Take an appropriate amount of sample 1 and place it in five 10mL volumetric flasks. Add 50, 100, 150, 200, and 250μL of 100mg / L copper standard solution in sequence. Then dilute to 10mL with sample 1, mix well, and let it stand for more than 4h to obtain samples 2–6 with copper ion concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5mg / L, respectively, and cyanide concentrations of 500μg / L.

[0054] 2.2 Pre-processing method Pretreatment method 1: Process according to GB5009.36-2023 33.1.2 (add 1.00 mL of sample to a 25 mL volumetric flask, add 1.0 g / L NaOH to the volume, and let it stand for 10 minutes).

[0055] Pretreatment method 2: Accurately pipette 1.00 mL of sample into a 25 mL volumetric flask, add 5 mL of 0.5 g / L EDTA-2Na aqueous solution, and then dilute to the mark with 1.0 g / L NaOH aqueous solution, mix well, and let it stand for 10 min.

[0056] 2.3 Detection methods Detection method 1: Using the improved flow path ( Figure 2 ) and optimized parameters (same as Example 1④).

[0057] Detection method 2: Using the "dynamic online chelation" of the present invention to improve the flow path ( Figure 3 ) and optimized parameters (same as Example 1④).

[0058] 2.4 Results

[0059] Conclusion: Copper ions significantly interfere with cyanide detection; pre-chelation (pretreatment method 2) can partially suppress the interference, but cyanide is easily lost in an open environment; the "dynamic online chelation" system of the present invention can effectively eliminate interference when the copper ion concentration of the sample is ≤2 mg / L.

[0060] Example 3 3.1 Samples and spikes Sample 1: Luzhou-flavor liquor, copper ion 0.24 mg / L, take 10 mL, add 40 μL of 50 mg / L cyanide standard solution, mix well to obtain cyanide spiked to 200 μg / L.

[0061] Sample 2: health wine, copper ion 0.48 mg / L, same operation as above.

[0062] Sample 3: wine, copper ion 0.82 mg / L, same operation as above.

[0063] 3.2 Pretreatment and determination All were processed according to method ⑤ in Example 1, and then measured according to step B of method in Example 1.

[0064] 3.3 Results Sample 1: Cyanide 308.39 μg / L, spiked sample 498.72 μg / L, recovery rate 95.17%.

[0065] Sample 2: Cyanide 27.81 μg / L, spiked sample 211.34 μg / L, recovery rate 91.77%.

[0066] Sample 3: Cyanide 907.53 μg / L, spiked sample 1103.61 μg / L, recovery rate 98.04%.

[0067] Conclusion: The method of the present invention is applicable to liquor samples with different complex matrices, such as liquor, health wine, and wine, with spiked recoveries of 91.77%–98.04%, showing universal applicability.

[0068] At the same time, the above samples were tested using the detection method 1: using the improved flow path ( Figure 2 ) and optimized parameters (same as Example 1④). The results were as follows: Sample 1: cyanide 308.67 μg / L, spiked sample 462.45 μg / L, recovery rate 86.89%.

[0069] Sample 2: Cyanide 19.56 μg / L, spiked sample 182.18 μg / L, recovery rate 81.31%.

[0070] Sample 3: Cyanide 789.83 μg / L, spiked sample 908.23 μg / L, recovery rate 75.92%.

[0071] Conclusion: Compared with the previous method, the detection accuracy of the method for copper ions in samples is greatly improved.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the scope of the present invention should be included in the scope of protection of the present invention.

Claims

1. A flow injection dynamic chelation determination system for cyanide in wine, characterized in that: include: Flow injection analyzer (1), equipped with: A sample flow path (11) for conveying the wine sample to be tested; a reagent flow path (12) for conveying distillation reagents; a peristaltic pump (13), used to provide driving force for the sample flow path and the reagent flow path; The chelating agent flow path (2) is connected to the outlet end of the sample flow path (11) and the two inlet ends of the first three-way connector (3) respectively, and is used to inject the chelating agent solution into the wine sample so that the copper ions in the wine sample form a complex with the chelating agent and release cyanide; The second three-way connector (4) is connected to the outlet end of the first three-way structure (3) and the outlet end of the reagent flow path (12) respectively with the inlet end of the second three-way connector (4). The second three-way connector (4) is used to coaxially merge the mixed wine sample-chelating agent solution and the distilled reagent again; A detection unit (5) is used to perform cyanide determination on the liquid mixed by the second three-way connector (4); Temperature control unit (6) is used to heat the combined liquid to 80℃–130℃ before testing The first three-way connector (3), the second three-way connector (4), the sample flow path (11), the reagent flow path (12), the chelating agent flow path (2) and the connecting pipeline are all made of inert polymer pipes to form a closed flow path.

2. The system according to claim 1, wherein The inert polymer tubing is an FEP capillary or a Tygon tube, wherein the sample flow path (11) has an inner diameter of 0.6–1.3 mm and a total length of 20–120 cm; the chelating agent flow path (2) has an inner diameter of 0.6–1.3 mm and a total length of 50–120 cm; and the reagent flow path (12) has an inner diameter of 0.5–1.5 mm and a total length of 20–120 cm.

3. The system according to claim 1, wherein: The chelating agent is disodium ethylenediaminetetraacetate, the solution concentration of which is 0.1 g / L-1.0 g / L, and is degassed before use.

4. The system according to claim 1, wherein: The invention also includes a flow ratio control module for maintaining the volume flow ratio of the wine sample to the chelating agent solution at 4:1-5:1 in real time.

5. The system according to claim 1, wherein: The rotation speed of the peristaltic pump is 15-30 rpm.

6. The system according to claim 1, wherein: The temperature control unit (6) comprises: a first-level heating module (61) with a temperature of 120°C; a second-level heating module (62) with a temperature of 85°C, wherein the second heating module is arranged in series after the first heating module.

7. A method for determining cyanide in wine using the system according to any one of claims 1 to 6, characterized in that: The steps include: S1, inject the wine sample into the sample flow path; S2. Inject the degassed chelating agent solution into the chelating agent flow path and mix it with the wine sample at the first three-way joint for ≤5s to chelate the copper ions; S3, mixing the mixed solution obtained in S2 with the distillation reagent at the second three-way joint, and then heating it through a temperature control unit; S4, measuring the cyanide content of the heated liquid obtained in S3 by a detection unit; The upper limit of the anti-interference of the method for copper ions in wine samples is 2 mg / L.

8. The method according to claim 7, wherein The colorimetric detection adopts the isonicotinic acid-barbituric acid method and performs colorimetric determination at a wavelength of 600 nm.

9. The method according to claim 7, wherein The detection limit was ≤2 μg / L, the relative standard deviation was ≤2.5%, and the recovery rate was 90%–110%.

10. Use of the system according to any one of claims 1 to 6 or the method according to any one of claims 7 to 9 in determining the cyanide content in white wine, yellow wine, grape wine or health wine.

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