Integrated magnetic stirring headspace detection method for carrageenan solvent residues

Abstract

The invention discloses an integrated magnetic stirring headspace detection method for carrageenan solvent residues, and relates to the field of food additive analysis and detection. In order to solve the problems that carrageenan is difficult to disperse uniformly after standing, heating and swelling, and solvent residues are not completely extracted, the method comprises the following steps: putting a carrageenan sample, water and a magnetic stirrer into a headspace sampling bottle, sealing, magnetically stirring and heating at 80-100 DEG C for 45-75 minutes, and directly carrying out headspace gas chromatography analysis. According to the method, a'magnetic stirring heating-headspace 'integrated closed design is created for the first time, a national standard tedious distillation process is replaced, internal residues are released through cooperation of magnetic stirring and heating, and the extraction efficiency is remarkably improved. The method is easy and convenient to operate, high in precision, stable in recovery rate, free of special equipment, capable of reducing errors and safety risks and suitable for detection of methanol and isopropanol residues of carrageenan and similar difficult-to-dissolve colloids.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food additive analysis and detection, in particular to a method for detecting methanol and isopropyl alcohol solvent residues of carrageenan which swells and is difficult to disperse uniformly in water by using integrated magnetic stirring headspace technology, and the method is suitable for quality monitoring and safety detection of carrageenan products by food additive production enterprises and quality supervision and inspection institutions. BACKGROUND

[0002] As an important food hydrophilic colloid, carrageenan is widely used in food processing such as dairy products, meat products and jelly. In the production process, methanol and isopropyl alcohol are often used as solvents, and the residual amount of such solvents in the finished product is directly related to food safety. It is a key detection index required by the national standard GB / T 1886.169-2016 Food Safety National Standard Food Additives Carrageenan.

[0003] The current mainstream detection method has significant technical defects: the current national standard adopts a pretreatment process of distillation- condensation collection- transfer- constant volume- gas chromatography, which needs to weigh 5.0g of sample, consumes 200mL of pure water, and takes 120 minutes for distillation and multiple transfer and constant volume operations. The whole process takes more than 3 hours, which is not only complicated and inefficient, but also easy to cause loss or pollution of the target due to solvent evaporation, container adsorption and operation errors, with poor detection precision (RSD up to 4.35%), and there is a safety risk of explosion and scalding when the open distillation reaches 100℃. At the same time, the standard recommends using packed column gas chromatography, which has low separation efficiency and is difficult to effectively separate interfering substances in complex matrix, resulting in insufficient accuracy of trace residue detection.

[0004] Existing improvement schemes have not solved the core problems: for example, Chinese patent application CN201810767200.8 discloses a method for determining residual solvents in carrageenan. This method only optimizes the gas chromatography temperature program and improves the separation degree, but still uses the complicated pretreatment process of distillation- transfer, and does not fundamentally simplify the operation and reduce the error.

[0005] In summary, the existing technology has not solved the core contradiction between the "complex, open and inefficient pretreatment process" and the "efficient, accurate and safe modern detection demand". Therefore, it is urgent to propose an efficient detection method for carrageenan solvent residues to solve the above technical problems. SUMMARY

[0006] Therefore, the present application proposes an integrated magnetic stirring headspace detection method for carrageenan solvent residues, which seamlessly integrates magnetic stirring heating and headspace sampling technology in a single closed container, solving the technical problems of incomplete extraction, complicated process and large error in the prior art.

[0007] The technical scheme of the present application is implemented as follows: An integrated magnetically stirred headspace detection method for residual carrageenan solvent includes the following steps: preparing a series of gradient standard working solutions of methanol and isopropanol, and sealing them together with water and a magnetic stir bar in a headspace injection vial; detecting the solutions using a headspace gas chromatograph (HS-GC-FID) and plotting a standard curve; placing the carrageenan sample to be tested, water, and a magnetic stir bar in a headspace injection vial, sealing the vial, heating it with magnetic stirring, detecting the solutions using a headspace gas chromatograph, and calculating the residual solvent content based on the standard curve.

[0008] In some embodiments, the carrageenan sample to be tested, water, and a magnetic stir bar are placed together in a headspace vial, sealed, and then placed on a magnetic stirring and heating device, and stirred and heated at 80°C to 100°C for 45 to 75 minutes.

[0009] When carrageenan is statically heated with water, only the surface layer absorbs water and swells, while the inner layer remains a dense structure, making it difficult to fully release the encapsulated solvent. This invention selects a heating temperature of 80°C to 100°C, which allows the outer layer of carrageenan to expand and soften, making it easier to break down under the mechanical shear force of continuous magnetic stirring, promoting the diffusion of residual solvent, while ensuring the full volatilization of methanol (boiling point 64.7°C) and isopropanol (boiling point 82.4°C). The stirring and heating time of 45 to 75 minutes is based on the diffusion kinetics of residual carrageenan solvent, ensuring that the bound solvent fully diffuses into the headspace phase, avoiding insufficient extraction or excessive extraction time.

[0010] In some embodiments, the heating temperature is 90°C.

[0011] In some embodiments, the stirring and heating time is 60 minutes.

[0012] Experiments with different time gradients verified that solvent residue extraction tended to reach equilibrium at 45 min (methanol recovery rate 95%), and complete extraction could be achieved at 60 min (recovery rate 98%). Extending to 75 min did not significantly improve the recovery rate. The choice of 60 min ensured both thorough extraction and detection efficiency, avoiding unnecessary time consumption.

[0013] In some embodiments, the mass-to-volume ratio of the carrageenan sample to water is 1 g:(40~60) mL, preferably 1 g:40 mL.

[0014] The liquid-to-solid ratio is designed based on the synergistic effect of headspace vial volume and carrageenan foaming characteristics. A range of 1 g:(40~60) mL ensures that the liquid volume is sufficient to fully wet and disperse the carrageenan particles, while reserving ≥60% headspace space to provide a stable and sufficient phase equilibrium environment for the evaporation of solvent residues. The preferred value of 1 g:40 mL achieves the optimal balance between dispersion effect and detection sensitivity. When the liquid-to-solid ratio is lower than 1:40, the water volume is insufficient, resulting in incomplete sample dispersion and reduced extraction efficiency. When the ratio is higher than 1:60, the headspace concentration is too low, which will reduce the detection sensitivity.

[0015] In some embodiments, the headspace sampler conditions of the headspace gas chromatograph are: equilibration time 15-30 min, heating temperature 80-95 °C, injection loop temperature 90 °C, transfer line temperature 100 °C, and gas phase circulation time 24 min, preferably equilibration time 20 min and heating temperature 80 °C.

[0016] Headspace equilibration is a crucial step in achieving equilibrium for the transfer of the target substance from the liquid phase to the gas phase. An equilibration time of 15–30 min ensures stable concentration of the target substance in the headspace gas phase. An equilibration temperature of 80–95 °C works synergistically with the initial stirring and heating temperature to prevent redissolution of the target substance. The combination of an equilibration time of 20 min and an equilibration temperature of 80 °C is the optimal parameter verified by experiments, ensuring sufficient equilibration while avoiding solvent evaporation or concentration fluctuations caused by excessively long equilibration times.

[0017] In some embodiments, the gas chromatographic conditions of the headspace gas chromatograph include: a CP-WAX57CB capillary column and a carrier gas of high-purity nitrogen with a purity ≥99.999%.

[0018] The CP-WAX57CB capillary column is a highly polar chromatographic column with high polarity matching with methanol and isopropanol, which can effectively separate interfering substances in complex matrices. Compared with the packed column of the national standard method, it has a higher theoretical plate number (>20000 / m) and the separation efficiency is improved by 10-20 times. High-purity nitrogen with a purity of ≥99.999% can avoid the interference of impurities in the carrier gas on the detector, ensure the stability of the detection signal, and improve the accuracy of trace residue detection.

[0019] In some embodiments, the gas chromatography conditions further include: an injection port temperature of 190°C, split injection with a split ratio of 5:1, constant flow mode, and a column flow rate of 1.2 mL / min.

[0020] The 190℃ injection port temperature ensures rapid vaporization of the target analyte in the headspace phase, avoiding condensation and adsorption; the 5:1 split ratio controls the injection volume, preventing column overload and peak tailing; the 1.2mL / min constant flow mode ensures stable linear flow rate of the target analyte within the column, improves retention time repeatability, and provides assurance for accurate quantification.

[0021] In some embodiments, the column oven program for the gas chromatograph is as follows: initial temperature 40°C held for 3 min, temperature increased to 70°C at a rate of 5°C / min, then increased to 180°C at a rate of 15°C / min and held for 5 min, and finally increased to 200°C at a rate of 20°C / min and held for 3 min.

[0022] This temperature program is designed to address the boiling point differences and matrix interference between methanol and isopropanol. An initial temperature of 40°C for 3 minutes allows for preliminary separation of the target analyte from low-boiling-point impurities. A slow heating rate of 5°C / min effectively distinguishes methanol (retention time 6.569 min) from isopropanol (retention time 7.226 min). A rapid subsequent temperature rise to 200°C and hold thoroughly flushes out residual high-boiling-point impurities from the column, preventing cross-contamination and ensuring detection stability.

[0023] In some embodiments, the magnetic stir bar is a 4*13mm glass magnetic stir bar; the detector of the headspace gas chromatograph is a flame ionization detector (FID) with a detector temperature of 220°C.

[0024] The 4*13mm size is compatible with the volume of a 20mL headspace vial. The glass material has good chemical stability and will not react with the sample or solvent. The FID detector has high sensitivity to carbon-containing organic compounds, and the detector temperature of 220℃ ensures that the target analyte is fully combusted, thereby improving the detection signal intensity and meeting the detection requirements for trace solvent residues.

[0025] In some embodiments, the solvent residue includes methanol and / or isopropanol.

[0026] Methanol and isopropanol are the most commonly used solvents in the production of carrageenan and are also key residual indicators that are required to be tested according to national standards. This invention specifically optimizes the extraction and detection conditions of these two solvents to ensure that the test results meet the requirements of national standards.

[0027] The present invention has the following advantages over the prior art: (1) This invention completely abandons the traditional complex open operation mode that relies on distillation, transfer, and volume adjustment. It innovatively integrates "magnetic stirring heating extraction" and "headspace sampling" technology seamlessly in a single closed container, realizing a "one-step" sample pretreatment. This not only shortens the pretreatment time, but also greatly reduces the error risk introduced by multi-step operations, making the method particularly suitable for the rapid detection of batch samples.

[0028] (2) This invention addresses the industry pain point that solid particles such as carrageenan, which are difficult to dissolve in water, tend to "swell" and become difficult to disperse evenly when heated under static conditions. It introduces magnetic stirring heating technology, which uses continuous mechanical shear force to forcibly break down the particles and promote diffusion. This dynamic extraction process accelerates the swelling of carrageenan particles and the diffusion rate of the internal solvent, thereby shortening the time required for extraction equilibrium. This allows for the efficient and complete release of bound solvent residues embedded within the sample. Experiments have shown that this method has a significantly higher extraction efficiency for actual process residues than static heating methods.

[0029] (3) The capillary column gas chromatography technology used in this invention, in synergy with the innovative pretreatment method, provides separation efficiency, sensitivity, and resolution far superior to the national standard packed column method. It can effectively separate interfering substances in complex matrices, ensuring that target substances such as methanol and isopropanol obtain characteristic chromatographic peaks with sharp peak shapes and stable retention times, greatly improving the reliability and accuracy of trace residue detection.

[0030] (4) Compared with the open distillation operation of heating water to boiling in the national standard method, the present invention only performs low-temperature (e.g., 90°C) heating and stirring in a closed headspace flask, eliminating safety risks such as scalding and boiling over, making the pretreatment process safer and more controllable. At the same time, the present invention does not require special expensive equipment and can be implemented using conventional magnetic stirring heaters and headspace gas chromatographs, which significantly reduces the detection cost of laboratories and the technical threshold of operators, and is conducive to its widespread application in testing institutions at all levels. Attached Figure Description

[0031] 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.

[0032] Figure 1 This is a standard curve diagram of methanol in Example 1 of the present invention; Figure 2 This is a standard curve of isopropanol in Example 1 of the present invention; Figure 3 The chromatograms of methanol and isopropanol in carrageenan standard sample in Example 1 of this invention are shown below. Figure 4 The chromatograms of methanol and isopropanol in the carrageenan sample of Example 2 of this invention are shown below. Figure 5 The chromatograms of methanol and isopropanol obtained by the national standard distillation method of carrageenan in Comparative Example 1 of this invention are shown below. Figure 6The chromatograms of methanol and isopropanol obtained by static heating of carrageenan in Comparative Example 2 of this invention are shown below. Figure 7 This is a comparison diagram of magnetic stirring heating (left) and static heating (right) of carrageenan in Comparative Example 2 of the present invention. Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1 This embodiment provides an integrated magnetic stirring headspace detection method for carrageenan solvent residue, including the following steps: Step 1: Preparation of Experimental Materials and Instruments. The sample used was commercially available food additive carrageenan sample 1; reagents were chromatographically pure methanol and isopropanol, and the experimental water was ultrapure water; the instruments and equipment included an Agilent 7890B gas chromatograph equipped with a flame ionization detector (FID), an Agilent 7697A headspace autosampler, and a magnetic stirring device with heating function; consumables included 20 mL headspace vials, sealed caps with PTFE / silicone gaskets, and 4*13mm glass magnetic stir bar.

[0035] Step 2: Preparation of standard solutions. Accurately weigh appropriate amounts of methanol and isopropanol, place them in 10 mL volumetric flasks, dissolve and dilute to volume with pure water to prepare stock solutions for each standard. Then, pipette appropriate volumes of each stock solution into 25 mL volumetric flasks, dilute and dilute to volume with pure water, and mix thoroughly to prepare mixed standard solutions. Accurately pipette 0.10, 0.20, 0.50, 1.00, and 2.00 mL of the mixed standard solution, and add pure water to bring the solvent to 10 mL to prepare a series of mixed standard test samples with a concentration gradient of 1–20 mg / L.

[0036] Step 3: Sample pretreatment. Weigh 0.25 g (accurate to 0.0001 g) of carrageenan sample 1 and place it in a 20 mL headspace vial. Add 10 mL of pure water and a 4*13 mm glass magnetic stir bar. After sealing the vial, stir magnetically at 90℃ for 60 min.

[0037] Step 4: Instrument analysis conditions settings. Headspace conditions: equilibration time 20 min, heating temperature 80℃, injection loop temperature 90℃, transfer line temperature 100℃, gas circulation time 24 min, pressurization time 0.25 min, injection time 0.5 min; Gas chromatography conditions: CP-WAX 57CB capillary column, carrier gas is high-purity nitrogen with a purity ≥99.999%, split ratio 5:1, column flow rate 1.2 mL / min, injection port temperature 190℃, temperature program is: initial temperature 40℃ held for 3 min, increased to 70℃ at a rate of 5℃ / min, then increased to 180℃ at a rate of 15℃ / min and held for 5 min, finally increased to 200℃ at a rate of 20℃ / min and held for 3 min; detector temperature is 220℃.

[0038] Step 5: Determination of standard curve, limit of detection, and limit of quantitation. Plot a standard curve with the peak areas of methanol and isopropanol in a series of standard solutions on the ordinate and the corresponding concentrations on the abscissa, as shown below. Figures 1-2 As shown in Table 1, the limit of detection (LOD) was calculated based on a signal strength / baseline noise ratio (S / N) of 3:1, and the limit of quantitation (LOQ) was calculated based on a S / N ratio of 10:1.

[0039] Step 6: Sample Determination. The sample to be tested and the standard solution are tested under the same detection conditions, such as... Figure 3 As shown, the peak areas of methanol and isopropanol are substituted into the standard curve to calculate the mass concentration of the corresponding solvent residue in the sample.

[0040] Step 7: Repeatability test. Take 6 samples to be tested, process and detect them according to the methods in Steps 3 and 6, record the peak areas of methanol and isopropanol, and calculate the relative standard deviation (RSD) of the peak areas. The results are shown in Table 2.

[0041] Step 8: Accuracy Experiment. Carrageenan samples with low background values ​​were selected for spiked recovery experiments at low, medium, and high concentration levels. Three parallel samples were set up for each concentration. The recovery rates of methanol and isopropanol were calculated, and the results are shown in Table 3.

[0042] Table 1: Standard Curve Parameters for Methanol and Isopropanol

[0043] Table 2: Repeatability test data of methanol and isopropanol in the test samples

[0044] Table 3: Experimental Results of Accuracy of Methanol and Isopropanol in Carrageenan 1

[0045] Example 2 This embodiment provides an integrated magnetic stirring headspace detection method for carrageenan solvent residue, including the following steps: Step 1: Preparation of experimental materials and instruments. The sample used was commercially available food additive carrageenan sample 2; reagents, instruments, and consumables were the same as in Example 1.

[0046] Step 2: Preparation of standard solution. The preparation method is the same as in Example 1.

[0047] Step 3: Sample pretreatment. Weigh 6 portions of carrageenan sample 2, each 0.25 g (accurate to 0.0001 g), and place them in 6 20 mL headspace vials. Add 10 mL of pure water and a 4*13 mm glass magnetic stir bar to each vial. After sealing, stir magnetically at 90 °C for 10 min, 20 min, 30 min, 45 min, 60 min, and 75 min, respectively.

[0048] Step 4: Instrument analysis conditions settings. These are the same as those in Example 1.

[0049] Step 5: Sample Determination. Six treated samples were tested under the same detection conditions. The concentrations of methanol and isopropanol in each sample were calculated. The results are shown in Table 4 and... Figure 4 As shown.

[0050] Table 4: Experimental results of different magnetic stirring heating times

[0051] Comparative Example 1 This comparative example provides a method for detecting residual carrageenan solvent, comprising the following steps: Step 1: Preparation of experimental materials and instruments. The sample used was commercially available carrageenan sample 2, the same as in Example 2; the reagents were chromatographically pure methanol, isopropanol, ultrapure water, and silicone defoamer; the instruments and equipment included a 500 mL round-bottom distillation flask, a condenser, a receiving flask, a 200 mL volumetric flask, and a gas chromatograph configured as in Example 1.

[0052] Step 2: Experimental Procedure. Strictly follow the procedures specified in the national standard GB / T 1886.169-2016: Accurately weigh three 5.0 g carrageenan samples and place them in three separate 500 mL round-bottom distillation flasks. Add 200 mL of ultrapure water and 1 mL of defoamer to each flask, shake to mix, and then connect to the distillation apparatus for distillation. Use a 200 mL volumetric flask pre-filled with water as the receiver to distill approximately 100 mL of distillate. Remove the volumetric flask, cool to room temperature, and then add water to the mark and mix well. Accurately transfer 8.0 mL of the distillate to a 20 mL headspace vial, add 1.0 mL of internal standard solution, and seal the vial tightly.

[0053] Step 3: Instrument analysis conditions setup. Headspace and gas chromatography conditions were set consistent with those in Example 1 to ensure fair comparison.

[0054] Step 4: Sample Determination. Three samples were tested under the set conditions. The concentrations and RSD values ​​of methanol and isopropanol were calculated, and the results were compared with those obtained using the 60-minute magnetic stirring and heating method in Example 2. The results are shown in Table 5. Figure 5 As shown.

[0055] Table 5: Experimental results (mg / kg) of the national standard method and the method of this invention

[0056] Comparative Example 2 This comparative example provides a method for detecting residual carrageenan solvent, comprising the following steps: Step 1: Preparation of experimental materials and instruments. The sample used was commercially available carrageenan sample 2, the same as in Example 2; the reagents, instruments, equipment, and consumables were all the same as in Example 1.

[0057] Step 2: Preparation of standard solution. The preparation method is the same as in Example 1.

[0058] Step 3: Sample pretreatment. Weigh 0.25 g (accurate to 0.0001 g) of carrageenan sample 2 into 3 20 mL headspace vials, add 10 mL of pure water and a 4*13 mm glass magnetic stir bar to each, seal the vials, and place them in an oven at 90 °C for 60 min (as control group); at the same time, take 3 samples treated in the same way as in Example 2 and heated with magnetic stirring (as experimental group).

[0059] Step 4: Instrument analysis conditions settings. These are the same as those in Example 1.

[0060] Step 5: Sample Determination. The control and experimental group samples were tested under the same detection conditions. The concentrations of methanol and isopropanol, and their RSD values ​​were calculated. The results are shown in Table 6 and... Figure 6 As shown.

[0061] Figure 7 The comparative example also shows a comparison of the physical morphology of carrageenan after magnetic stirring and heating (left) and static heating (right). It can be clearly seen from the figure that carrageenan is prone to "swelling" and is difficult to disperse evenly when statically heated. However, the introduction of magnetic stirring and heating technology promotes the uniform diffusion of carrageenan through continuous mechanical shear force.

[0062] Table 6: Experimental results (mg / kg) of the static heating method and the method of the present invention

[0063] Example 3 The purpose of this experiment is to verify the synergistic effect of heating temperature, stirring time, and liquid-to-solid ratio, and to prove the uniqueness and optimality of the preferred parameter combination of the present invention. Specifically, it includes the following steps: Step 1: Preparation of experimental materials and instruments. The sample used is the same carrageenan sample 1 as in Example 1; the reagents, instruments, equipment, and consumables are all the same as in Example 1.

[0064] Step 2: Preparation of standard solution. The preparation method is the same as in Example 1.

[0065] Step 3: Experimental Design. A three-factor, three-level orthogonal experimental design was adopted. Factor A was the heating temperature (80℃, 90℃, 100℃), Factor B was the stirring time (45min, 60min, 75min), and Factor C was the liquid-to-solid ratio (1g:30mL, 1g:40mL, 1g:50mL). There were a total of 9 groups of experiments, and 3 parallel samples were set up for each group of experiments.

[0066] Step 4: Sample pretreatment. According to the orthogonal experimental design, weigh the corresponding mass of carrageenan sample and place it in a 20mL headspace vial. Add the corresponding volume of pure water and a 4*13mm glass magnetic stir bar. After sealing the vial, stir magnetically for a set time at the set temperature.

[0067] Step 6: Instrument analysis conditions settings. These are the same as those in Example 1.

[0068] Step 7: Performance Verification. Test each sample group and record the recovery rates of methanol and isopropanol; analyze the peak tailing factor using a chromatography workstation (≤1.2 is acceptable); observe the sample solution state to determine if degradation exists (no turbidity, no precipitation indicates no degradation); calculate the comprehensive score (out of 100) based on the weights of "recovery rate 60 points, peak tailing factor 30 points, sample stability 10 points," and the results are shown in Table 7.

[0069] Table 7: Experimental Results of Orthogonal Optimization of Parameters

[0070] Example 4 The experimental objective of this embodiment is to verify the synergistic effect of the pretreatment and chromatographic conditions of the present invention, and to demonstrate the targeted optimization of the chromatographic conditions. Specifically, it includes the following steps: Step 2: Preparation of experimental materials and instruments. The sample used was carrageenan sample 1 after pretreatment in Example 1; the reagents and instruments were the same as in Example 1 except for the chromatographic column. In addition, an HP-INNOWAX capillary column and a national standard recommended packed column were prepared (specific model: polyethylene glycol PEG-20M stationary phase packed column, column length 3m, inner diameter 3mm, packing material is silanized diatomaceous earth carrier, stationary phase coating amount 10%, packing material particle size 0.18-0.25mm).

[0071] Step 2: Preparation of standard solution. The preparation method is the same as in Example 1.

[0072] Step 3: Experimental Design. Nine sets of experiments were designed, using different column types (CP-WAX 57CB capillary column, HP-INNOWAX capillary column, and nationally recommended packed column) and different temperature programs (the program of this invention, the conventional isothermal program, and the temperature program of CN201810767200.8).

[0073] Step 4: Instrument analysis conditions settings. Except for the column type and temperature program, the other conditions are the same as in Example 1; the standard isothermal program is 70°C for 20 minutes; the temperature program of CN201810767200.8 is set according to the parameters disclosed in its patent.

[0074] Step 5: Performance Verification. Test each sample group and record the resolution (≥1.5 for acceptable) and retention time RSD values ​​of methanol, isopropanol, and impurity peaks; calculate the detection limits for methanol and isopropanol. The results are shown in Table 8.

[0075] Table 8: Results of the synergistic validation experiment of chromatographic conditions

[0076] Example 5 The purpose of this experiment is to compare the dispersion effects of ultrasound, microwave, and magnetic stirring, and to demonstrate the unique advantages of the dispersion method of this invention. Specifically, it includes the following steps: Step 1: Preparation of experimental materials and instruments. The sample used is carrageenan sample 2, which is the same as in Example 2; the reagents and instruments are the same as in Example 1, except that an ultrasonic extractor and a microwave extractor are added; the consumables are the same as in Example 1.

[0077] Step 2: Preparation of standard solution. The preparation method is the same as in Example 1.

[0078] Step 3: Experimental Design. Three experimental groups were set up, with three parallel samples in each group: Control group 1 was ultrasonic + headspace (headspace vial placed in ultrasonic instrument, ultrasonicated at 90℃ for 60 min); Control group 2 was microwave + headspace (headspace vial placed in microwave extractor, microwaved at 90℃ for 60 min, power 300W); Experimental group was the method of this invention (magnetic stirring at 90℃ for 60 min).

[0079] Step 4: Sample pretreatment. The sample amount, water volume, and headspace vial specifications were the same for all three groups of experiments, with the only difference being the dispersion method. The specific pretreatment was carried out according to the conditions of the corresponding group.

[0080] Step 5: Instrument analysis conditions settings. These are the same as those in Example 1.

[0081] Step 6: Performance Verification. The three groups of samples were tested, and the detection concentrations and RSD values ​​of methanol and isopropanol were calculated. The market prices of the equipment required for each group were calculated, and the results are shown in Table 9.

[0082] Table 9: Comparative Experimental Results of Different Dispersion Methods + Headspace Sampling

[0083] The experimental results of each embodiment and comparative example fully verify the scientific nature and superiority of the detection method of the present invention. Example 1, through standard curve, repeatability, and accuracy experiments, confirms that the method has a good linear relationship (R0). 2 With a concentration ≥0.9999, excellent precision (RSD≤4.21%), and stable recovery rate (92.8%-101%), this invention meets the requirements for trace residue detection. Example 2 clarifies that 45-60 minutes is the optimal stirring and heating time, achieving a balance between extraction efficiency and detection speed. Examples 3-4 further verify the synergistic optimization effect of temperature, time, and liquid-solid ratio. Comparison Example 1, compared with the national standard method, shows that this invention replaces the cumbersome distillation process with a "one-step method," significantly improving precision (RSD reduced from 4.35% to 2.66%), shortening the detection cycle, and reducing safety risks while ensuring the consistency of detection results. Comparative Example 2 confirms that magnetic stirring is the key to overcoming the bottleneck of carrageenan swelling and improving extraction efficiency (methanol detection concentration increased by 3.8 times). Example 4 shows that the magnetic stirring method selected in this invention has irreplaceable advantages compared with other conventional methods.

[0084] In summary, the method of the present invention is simple to operate, accurate, reliable, and cost-controllable. It is suitable for the efficient detection of solvent residues in carrageenan and similar food colloids, and has broad application value.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An integrated magnetic stirring headspace detection method for carrageenan solvent residue, characterized in that, Includes the following steps: (1) Prepare a series of gradient standard working solutions of methanol and isopropanol. Place the series of gradient standard working solutions, water and magnetic stir bar together in a headspace injection bottle and seal it. Use a headspace gas chromatograph to detect the peak areas of methanol and isopropanol. (2) Plot a standard curve with the peak areas of methanol and isopropanol as the ordinate and the contents of methanol and isopropanol in a series of gradient standard working solutions as the abscissa. (3) Place the carrageenan sample to be tested, water and magnetic stir bar together in a headspace vial and seal it. Place it on a magnetic stirring and heating device and stir and heat at 80°C to 100°C for 45 min to 75 min to release the solvent residue in the sample particles. (4) The sealed headspace vial from step (3) was analyzed by headspace gas chromatography to obtain the peak areas of methanol and isopropanol; (5) Substitute the peak area from step (4) into the standard curve from step (2) to calculate the methanol and isopropanol content in the carrageenan to be tested; The mass-to-volume ratio of the carrageenan sample to water is 1 g:(40~60) mL, and the solvent residue includes methanol and / or isopropanol.

2. The integrated magnetic stirring headspace detection method for carrageenan solvent residue according to claim 1, characterized in that, The heating temperature in step (3) is 90°C.

3. The integrated magnetic stirring headspace detection method for carrageenan solvent residue according to claim 1, characterized in that, The stirring and heating time in step (3) is 60 minutes.

4. The integrated magnetic stirring headspace detection method for carrageenan solvent residue according to claim 1, characterized in that, The mass-to-volume ratio of the carrageenan sample to water was 1 g: 40 mL.

5. The integrated magnetic stirring headspace detection method for carrageenan solvent residue according to claim 1, characterized in that, The headspace sampler conditions for the headspace gas chromatograph are as follows: equilibration time 15-30 min, heating temperature 80-95℃, injection loop temperature 90℃, transfer line temperature 100℃, and gas phase circulation time 24 min.

6. The integrated magnetic stirring headspace detection method for carrageenan solvent residue according to claim 5, characterized in that, The headspace sampler has an equilibration time of 20 minutes and a heating temperature of 80°C.

7. The integrated magnetic stirring headspace detection method for carrageenan solvent residue according to claim 1, characterized in that, The gas chromatography conditions of the headspace gas chromatograph include: the chromatographic column is a CP-WAX57CB capillary column, and the carrier gas is high-purity nitrogen with a purity ≥99.999%.

8. The integrated magnetic stirring headspace detection method for carrageenan solvent residue according to claim 7, characterized in that, The gas chromatography conditions also include: an injection port temperature of 190°C, split injection with a split ratio of 5:1, constant flow mode, and a column flow rate of 1.2 mL / min.

9. The integrated magnetic stirring headspace detection method for carrageenan solvent residue according to claim 7, characterized in that, The column oven program for the gas chromatograph is as follows: initial temperature 40℃ held for 3 min, temperature increased to 70℃ at a rate of 5℃ / min, then increased to 180℃ at a rate of 15℃ / min and held for 5 min, and finally increased to 200℃ at a rate of 20℃ / min and held for 3 min.

10. The integrated magnetic stirring headspace detection method for carrageenan solvent residue according to claim 1, characterized in that, The magnetic stir bar is a 4*13mm glass magnetic stir bar; the detector of the headspace gas chromatograph is a flame ionization detector with a detector temperature of 220℃.

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