A flow injection dynamic chelation assay system for cyanide in wine

Abstract

This invention discloses a flow injection dynamic chelation assay system for cyanide in alcoholic beverages, used to eliminate the interference of copper ions on cyanide detection online. The system includes a flow injection analyzer with a sample flow path, a reagent flow path, and a peristaltic pump. The chelating agent flow path is coaxially connected to the sample flow path via a first three-way connector, allowing the chelating agent to complex with copper ions in the alcoholic sample in real time, releasing cyanide. The mixture is then connected to a distillation reagent via a second three-way connector, and after two-stage heating by a temperature control unit, the sample is colorimetrically determined by a detection unit. This assay system and method are applicable to complex matrices such as baijiu, huangjiu, wine, and health wine, requiring no additional hardware modifications, and offering low cost and high accuracy.

Description

Technical Field

[0001] This utility model relates to the field of detection and analysis technology, and specifically proposes a flow injection dynamic chelation determination system for cyanide in wine. Background Technology

[0002] Cyanide is one of the key toxic substances in the safety control of alcoholic beverages, and its accurate quantitative detection is crucial for controlling the quality and safety of alcoholic products and protecting consumer health. 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 testing, the interference from the complexity of the alcoholic sample matrix is ​​easily overlooked—especially the presence of copper ions (Cu) in the alcoholic beverage. 2+ (Health wines, baijiu, wines, huangjiu, etc., may introduce cyanide due to their raw materials, production processes, and storage methods.) - The formation of complexes may prevent cyanide from being released during distillation or hinder the colorimetric reaction, resulting in significantly lower detection results. This experimental study shows that when copper ion standard solution was added to 1.0 mg / L of freshly distilled strong-aroma baijiu (cyanide content was 406.57 μg / L, copper was not detected), mixed well, and allowed to stand for 4 hours, the cyanide content was detected according to the fifth method of GB5009.36-2023, and the detection result was only 69.15 μg / L.

[0003] The interference of copper ions in the conventional flow injection method for analyzing cyanide in wine is highly insidious, specifically in the following ways: ① Complex interference mechanism: The complexation equilibrium between copper ions and cyanide is affected by multiple factors, including the pH value and concentration of the wine sample, making it difficult to completely eliminate through conventional dilution. Furthermore, excessive dilution can lead to significant fluctuations in cyanide detection results. ② Risk of false negatives: Distorted cyanide detection results can easily be misjudged as "low cyanide content" wine samples, masking actual safety hazards. ③ Method limitations: Current national standard methods and literature reports do not provide effective solutions for the interference of copper ions in the flow injection analysis of cyanide.

[0004] Therefore, there is an urgent need for a measurement system and method that is simple in structure, highly compatible, and capable of eliminating copper ion interference in real time. Summary of the Invention

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

[0006] This experimental study found that the binding ability of cyanide ions to copper ions in wine samples is weaker than that of ethylenediaminetetraacetic acid (EDTA) to copper ions. Based on this principle, this invention effectively solves the problem of copper ion interference in cyanide flow injection analysis of wine by innovatively designing a "dynamic online chelation" flow path system. The main advantages of this invention are: ① For the first time, it introduces disodium EDTA as a mobile phase based on traditional flow injection analysis, realizing online dynamic chelation of copper ions in the wine sample in the closed flow injection system, and precisely controlling the mixing ratio of the wine sample and the disodium EDTA solution, effectively eliminating the interference of copper ions in the detection of cyanide in the wine sample. ② This invention adopts a flow pipeline design and flow path improvement and optimization, requiring no major hardware modifications, and has extremely high compatibility with fully automated flow injection analyzers, without affecting the compatibility of the instrument control software.

[0007] The technical solution of this utility model is achieved as follows: This utility model 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] Chelating agent flow path: This path connects coaxially with the sample flow path outlet via a first tee connector, and is used to inject a degassed disodium ethylenediaminetetraacetate (EDTA-2Na) solution into the wine sample. This chelating agent has a much higher complexing ability with copper ions than with cyanide ions, and can instantly displace and release the 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] The second three-way connector: the wine sample-chelating agent mixture and the distillation reagent are coaxially combined again to form a homogeneous reaction system.

[0011] Temperature control unit: Equipped with a primary heating module (120℃) and a secondary heating module (85℃) to precisely control the distillation and colorimetric reaction temperatures, ensuring a full and stable reaction. The secondary heating module is connected in series after the primary heating module.

[0012] Closed-loop design: All flow paths and T-joints are made of inert polymer tubing, such as FEP capillaries or Tygon tubes, with strictly matched inner diameters. The sample flow path has an inner diameter of 0.6–1.3 mm and a total length of 20–120 cm. The chelating agent flow path has an inner diameter of 0.6–1.3 mm and a total length of 50–120 cm. The reagent flow path has an inner diameter of 0.5–1.5 mm and a total length of 20–120 cm, forming a closed system with no volatilization and no residue.

[0013] Peristaltic pump speed setting: Controlled between 15–30 rpm, balancing response time and detection sensitivity.

[0014] Specifically, The flow injection analyzer is equipped with: Sample flow path, used to transport the wine sample to be tested; Reagent flow path, used to deliver distilled reagents; A peristaltic pump is used to provide driving force for the sample flow path and the reagent flow path; The chelating agent flow path is connected to the two inlet ends of the first three-way connector, respectively, at the outlet end of the sample flow path. It 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 connects the outlet end of the first three-way structure and the outlet end of the reagent flow path to the inlet end of the second three-way connector. The second three-way connector is used to coaxially combine the mixed wine sample-chelating agent solution and the distillation reagent again. The detection unit is used to determine the cyanide content in the liquid after it has been mixed through the second three-way connector. The temperature control unit is used to heat the combined liquids to 80℃–130℃ before detection. The first tee connector, the second tee connector, the sample flow path, the reagent flow path, the chelating agent flow path, and the connecting pipe are all made of inert polymer tubing, forming a closed flow path.

[0015] This utility model also provides a determination method, including the following steps: S1 Sample Injection: After national standard pretreatment, the wine sample is directly injected into the sample flow path without any additional offline steps.

[0016] S2 dynamic chelation: The degassed EDTA-2Na solution (concentration 0.1–1.0 g / L) is coaxially combined with the wine sample in the first three-way connector. The mixing time is ≤5s, copper ions are complexed in real time, and cyanide is completely released.

[0017] S3 Distillation-Color Development: After the mixture and distillation reagent are combined at the second three-way connector, they undergo a first-stage distillation at 120℃ and a second-stage color development at 85℃ to ensure that cyanide is efficiently converted into hydrogen cyanide.

[0018] S4 colorimetric determination: The classic isonicotinic acid-barbituric acid method was used to perform colorimetric determination at a wavelength of 600 nm. The entire process was automatically recorded and the results were output by the flow injection optical path system.

[0019] In some embodiments, the technical performance of the above-described measurement method is as follows: Upper limit of interference resistance: When the copper ion concentration is ≤2mg / L, the deviation of cyanide determination results is <5%.

[0020] Detection limit: ≤2μg / L, significantly better than the current standard.

[0021] Precision: Relative standard deviation (RSD) ≤ 2.5%.

[0022] Recovery rate: 90%–110%, covering complex matrices such as baijiu, huangjiu, wine, and health wine.

[0023] Detection throughput: 15 samples / hour, meeting the needs of batch testing.

[0024] The present invention has the following advantages over the prior art: Compared with existing technologies, this novel method for cyanide flow injection analysis of alcoholic beverages features: First, it pioneers a "dynamic online chelation" system, introducing a disodium ethylenediaminetetraacetate chelating solution as the mobile phase to achieve real-time chelation of copper ions in a closed flow path (avoiding cyanide loss from pre-chelation in open systems). Second, its innovative tubing design ensures thorough dynamic mixing of the chelating agent and the alcohol sample while precisely controlling the mixing ratio. Third, the improved flow path offers strong adaptability, achievable simply through adjustments to the flow tubing specifications and flow path modifications. Fourth, the optimized instrument parameters significantly improve the signal-to-noise ratio, enabling the method to resist copper ion interference up to an upper limit of 2 mg / L. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of the connection relationship of the flow injection dynamic chelation determination system for cyanide in wine according to this utility model. Figure 2 This is a flow path diagram for a cyanide flow injection apparatus in the prior art; Figure 3 This is a flow path diagram of the dynamic chelation determination system for cyanide in wine according to the present invention.

[0027] In the diagram: 1-Flow injection analyzer, 2-Chlorinating agent flow path, 3-First tee connector, 4-Second tee structure, 5-Detection unit, 6-Temperature control unit, 11-Sample flow path, 12-Reagent flow path, 13-Peristaltic pump, 61-First stage heating module, 62-Second stage heating module. Detailed Implementation

[0028] The technical solutions of this utility model will be clearly and completely described below with reference to the embodiments of this utility model. Obviously, the described embodiments are only a part of the embodiments of this utility model, and not all of them. Based on the embodiments of this utility model, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of this utility model.

[0029] It should be noted that when a component is referred to as being "fixed to" or "set on" another component, it can be directly on or indirectly on that other component. When a component is referred to as being "connected to" another component, it can be directly connected to or indirectly connected to that other component.

[0030] It should be understood that the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They 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, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0031] 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 technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0032] 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 the embodiments of this utility model pertain. If any definition stated in this section is contrary to or otherwise inconsistent with a definition stated in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definitions listed here shall prevail over those incorporated herein by reference.

[0033] Unless otherwise specified, the methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0034] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1 to 5” is disclosed, the described range should be interpreted as including ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, “1 to 3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range. In this specification and claims, range definitions may be combined and / or interchanged, unless otherwise stated, these ranges include all subranges contained therein.

[0035] In this embodiment, the flow injection instrument is a Beijing Jitian fully automated flow injection analyzer (model FF-QHWL08).

[0036] Example 1 Determination of cyanide in strong-aroma baijiu.

[0037] Instrument and System Setup Using a flow injection analyzer (1), its internal configuration is as follows: Sample flow path (11): FEP capillary inner diameter 0.80mm, length 110cm, Tygon pump tube inner diameter 1.30mm, length 38cm; Reagent flow path (12): Distillation reagent section FEP capillary inner diameter 0.60mm, length 110cm, Tygon pump tube inner diameter 0.76mm, length 38cm; Peristaltic pump (13): Set value 22 rpm (instrument panel reading); Chelating agent flow path (2): FEP capillary inner diameter 0.60mm, length 110cm, Tygon pump tube inner diameter 0.76mm, length 38cm, outlet end connected to the inlet end of the first tee connector (3); Second three-way connector (4): The outlet end of the first three-way connector (3) is coaxially connected with the outlet end of the reagent flow path (12); Temperature control unit (6): primary heating module 120℃, secondary heating module 85℃; Detection unit (5): Flow injection optical path system, wavelength 600nm.

[0038] All pipes and tees are made of inert polymer (FEP / Tygon) to form a closed flow path.

[0039] ① Reagent preparation 0.5g / EDTA-2Na chelate solution: Weigh 0.5g EDTA-2Na, dissolve in deionized water and bring the volume to 1000mL. Prepare fresh before use and degas by sonication for 15min.

[0040] Sodium hydroxide solution: 1.0 g / L, prepared according to GB5009.36-2023 31.2.1.

[0041] Distillation reagents, buffer solutions, chloramine T solution, and colorimetric reagents were all prepared in accordance with GB5009.36-2023 31.2.1.

[0042] Cyanide standard solution: Prepared according to GB5009.36-2023 31.4.

[0043] ② Pipeline assembly Sample tubes: FEP capillary inlet tube inner diameter 0.80mm×110cm, outlet tube inner diameter 0.80mm×22cm, intermediate Tygon pump tube inner diameter 1.30mm×38cm, connected in series with adapters.

[0044] Chelating agent solution mobile phase tube: FEP capillary inlet tube inner diameter 0.60mm×110cm, outlet tube inner diameter 0.60mm×52cm, intermediate Tygon pump tube inner diameter 0.76mm×38cm, connected in series with adapter.

[0045] The remaining piping is consistent with the original factory configuration.

[0046] ③ Flow path improvement The sample output tube and the EDTA-2Na output tube are mixed through the first tee connector (3).

[0047] The mixture is then mixed with the distillation reagent through the second three-way connector (4) for subsequent analysis.

[0048] ④ Parameter settings Peristaltic pump speed setting: 22 rpm Valve arrival time: 300s Injection time: 85s Sample cycle: 225s Injection time: 140s Cleaning time: 40s Needle cleaning time: 15s Primary heating module: 120℃ Secondary heating module: 85℃ Front-end acquisition gain: 110K ⑤ Sample selection and processing Take one batch of strong-aroma baijiu (copper ions 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 aqueous solution, mix well, and let stand for 10 min to obtain the sample to be tested.

[0049] ⑥ Sample testing Option A: The measurement was performed using the original flow path and the original parameters (peristaltic pump setting 20 rpm, injection time 80 s, cleaning time 30 s, primary heating 118 ℃, front-end acquisition gain 100 K).

[0050] Option B: Use the improved flow path and optimized parameters (see ④) for measurement.

[0051] ⑦ Results The same strong-aroma baijiu: The measured value under scheme A was 302.13 μg / L. The measured value using scheme B was 310.76 μg / L. The deviation between the two methods is only 2.82%, indicating that the improved flow path and parameters can be directly applied and are fully compatible with the instrument control software.

[0052] Example 2 2.1 Sample Preparation ① Prepare 100 mL of 50% vol ethanol aqueous solution (copper ions were not detected).

[0053] ② Take a portion of the solution into a 100mL volumetric flask, add 1mL of 50mg / L cyanide standard solution, and dilute to 100mL with ethanol aqueous solution. After mixing, sample 1 with a cyanide concentration of 500μg / L is obtained.

[0054] ③ Take an appropriate amount from sample 1 into five 10 mL volumetric flasks, add 50, 100, 150, 200, and 250 μL of 100 mg / L copper standard solution in sequence, then dilute to 10 mL with sample 1, mix well, and let stand for more than 4 hours to obtain samples 2–6 with copper ion concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 mg / L and cyanide concentrations of 500 μg / L.

[0055] 2.2 Preprocessing Method Pretreatment method 1: Treat according to GB5009.36-2023 33.1.2 (add 1.00mL of sample to a 25mL volumetric flask, add 1.0g / LNaOH to make up to volume, and let stand for 10min).

[0056] 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, then dilute to the mark with 1.0 g / L NaOH aqueous solution, mix well, and let stand for 10 min.

[0057] 2.3 Detection Methods Detection Method 1: Using the original flow path ( Figure 2 ) and optimized parameters (same as Example 1④).

[0058] Detection Method 2: Using the "Dynamic Online Chelation" improved flow path of this utility model ( Figure 3 ) and optimized parameters (same as Example 1④).

[0059] 2.4 Results

[0060] Conclusion: Copper ions significantly interfere with cyanide detection; pre-chelation (pretreatment method 2) can partially suppress the interference, but the open environment easily leads to the loss of cyanide; the "dynamic online chelation" system of this invention can effectively eliminate interference when the copper ion concentration in the sample is ≤2 mg / L.

[0061] Example 3 3.1 Samples and Spikes Sample 1: Strong-aroma baijiu, 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 200 μg / L.

[0062] Sample 2: Health wine, copper ion 0.48mg / L, same procedure as above.

[0063] Sample 3: Wine, copper ion concentration 0.82 mg / L, same procedure as above.

[0064] 3.2 Pretreatment and Determination All samples were processed according to method ⑤ in Example 1, and then measured according to step B of method 1 in Example 1.

[0065] 3.3 Results Sample 1: Cyanide 308.39 μg / L, spiked sample 498.72 μg / L, recovery rate 95.17%.

[0066] Sample 2: Cyanide 27.81 μg / L, spiked sample 211.34 μg / L, recovery rate 91.77%.

[0067] Sample 3: Cyanide 907.53 μg / L, spiked sample 1103.61 μg / L, recovery rate 98.04%.

[0068] Conclusion: The method of this invention is applicable to various complex matrix wine samples such as baijiu, health wine, and wine, with a spiked recovery rate of 91.77%–98.04%, demonstrating its universality.

[0069] Meanwhile, for the above samples, detection method 1 was adopted: using the improved flow path ( Figure 2 The optimized parameters (same as in Example 1④) were measured, and the results are as follows: Sample 1: Cyanide 308.67 μg / L, spiked sample 462.45 μg / L, recovery rate 86.89%.

[0070] Sample 2: Cyanide 19.56 μg / L, spiked sample 182.18 μg / L, recovery rate 81.31%.

[0071] Sample 3: Cyanide 789.83 μg / L, spiked sample 908.23 μg / L, recovery rate 75.92%.

[0072] Conclusion: Compared with the previous method, the method of this invention has a significant improvement in the detection accuracy of copper ions in samples.

[0073] The above description is only a preferred embodiment of the present utility model and is not intended to limit the present utility model. Any modifications, equivalent substitutions, improvements, etc., made within the concept of the present utility model should be included within the protection scope of the present utility model.

Claims

1. A flow injection dynamic chelation assay system for cyanide in wine, characterized by, include: The flow injection analyzer (1) is equipped with: Sample flow path (11) is used to transport the wine sample to be tested; Reagent flow path (12) is used to deliver distillation reagents; A peristaltic pump (13) is used to provide driving force for the sample flow path and the reagent flow path; The chelating agent flow path (2) and the outlet end of the sample flow path (11) are respectively connected to the two inlet ends of the first three-way connector (3) for injecting 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 outlet end of the first three-way connector (3) and the outlet end of the reagent flow path (12) are respectively connected to the inlet end of the second three-way connector (4). The second three-way connector (4) is used to coaxially combine the mixed wine sample-chelating agent solution and the distillation reagent again. The detection unit (5) is used to determine the cyanide content of the liquid after it has been mixed through the second three-way connector (4); Temperature control unit (6) is used to heat the combined liquid to 80℃–130℃ before detection. Among them, 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 pipe are all made of inert polymer tubing to form a closed flow path.

2. The system of claim 1, wherein, The inert polymer tubing is an FEP capillary or a Tygon tube. 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 of claim 1, wherein, The temperature control unit (6) includes a primary heating module (61) and a secondary heating module (62), with the secondary heating module (62) connected in series after the primary heating module (61).

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