A rapid visualization and fluorescence detection method for perchlorate in water and fresh tea leaves

Abstract

A method for rapid visualization and fluorescence detection of perchlorate in water and fresh tea leaves belongs to the field of detection technology. The method specifically comprises the following steps: (1) taking water and fresh tea leaves for pretreatment to obtain a test solution, and adding toluidine blue as a color indicator and fluorescent probe to the test solution; (2) the mixed solution of step (1) undergoes color development and fluorescence reaction, and after liquid-liquid extraction, the solution is allowed to stand, and colorimetry is performed to achieve rapid visualization detection of perchlorate in water and fresh tea leaves. The method has the advantages of simple operation, high sensitivity, short time consumption, low cost, good repeatability, etc., can complete the visualization detection of perchlorate in a relatively short time, facilitates on-site real-time monitoring, and provides technical support for the qualitative determination of perchlorate in water and fresh tea leaves. The fluorescence detection limits in water and fresh tea leaves are 0.15 μM and 0.36 mg / kg, respectively.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and in particular relates to a rapid visualization and fluorescence detection method for perchlorate in water and fresh tea leaves. Background Art

[0002] Perchlorate (ClO4 - ) is a thyroid toxin with high water solubility and high stability. It is frequently detected in aquatic environments and is a global pollutant. Perchlorate is enriched in plants through the water and soil environment, causing pollution of agricultural products and related foods. Tea is widely loved as a healthy drink at home and abroad, and its quality and safety are particularly important. So far, many researchers have found that the detection rate of perchlorate in tea is higher than 90%, and the perchlorate content in fresh tea leaves increases with maturity.

[0003] Existing detection technologies for perchlorate include ion chromatography (IC), liquid chromatography-mass spectrometry (LC-MS), surface enhanced Raman scattering (SERS), infrared absorption spectroscopy (IR), Raman spectroscopy, and capillary electrophoresis, etc. The above methods have low detection limits and high precision, but the site requirements for large instruments, the need for professional technicians to operate, and the lengthy pre-treatment process make them difficult to use for on-site real-time monitoring.

[0004] On November 22, 2019, the applicant applied for a method for rapid visual detection of perchlorate in water, tea soup and tea leaves. The publication number of the patent is CN 110940662 A. The method specifically includes the following steps: (1) taking water, tea soup and tea leaves for pretreatment to obtain a test solution, and adding methylene blue as a color indicator to the test solution; (2) adjusting the pH of the test solution obtained in step (1) to alkaline for color development reaction, and after liquid-liquid extraction, standing, and colorimetrically realizing rapid visual detection of perchlorate in water, tea soup and tea leaves. The invention has the advantages of simple operation, high sensitivity, short time consumption, low cost, and good repeatability. It can complete the visual detection of perchlorate in a short time, facilitate on-site real-time monitoring, and provide technical support for the qualitative determination of perchlorate in water, tea soup and tea leaves. The detection limits in water, tea soup and tea leaves can reach 0.005mM, 0.02mM and 5mg kg, respectively. -1 However, this method requires adjusting the pH of the sample solution to be alkaline; if the pH of the sample solution is unknown, a preliminary experiment is required to determine the amount of alkaline substance used to adjust the pH.

[0005] Therefore, it is particularly important to develop a simpler method for rapid visualization and fluorescence detection of perchlorate in water and fresh tea leaves that does not require adjustment of the detection environment (such as pH, ionic strength, etc.). Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for a method for rapid visualization and fluorescence detection of perchlorate in water and fresh tea leaves. The present invention uses toluidine blue as a color indicator and fluorescent probe, performs color development reaction and fluorescence sensitization under certain conditions, realizes rapid visualization and fluorescence detection of perchlorate in water and fresh tea leaves, and completes quantitative detection of trace perchlorate.

[0007] The present invention is specifically implemented by the following technical solutions:

[0008] A method for rapid visualization and fluorescence detection of perchlorate in water and fresh tea leaves, comprising the following steps:

[0009] 1) Take water and fresh tea leaves for pretreatment to obtain the test solution;

[0010] 2) Adding toluidine blue as a color indicator and a fluorescent probe to the test solution obtained in step 1) to react, after liquid-liquid extraction, the solution is allowed to stand, and the organic layer containing perchlorate appears blue, while the blank control organic layer appears red, thereby realizing rapid visual detection of perchlorate;

[0011] 3) Taking the organic layer for fluorescence detection to obtain the fluorescence intensity, substituting the value of the fluorescence intensity into the perchlorate standard curve to calculate the content of perchlorate in the sample to be tested.

[0012] Furthermore, the water pretreatment in step 1) is as follows: centrifuging the water sample and taking the supernatant.

[0013] Furthermore, the pretreatment of fresh tea leaves in step 1) is as follows: taking 2 g of fresh tea leaves, placing them in 10-50 mL of water or 0.1%-1% formic acid aqueous solution, ultrasonically extracting for 3-10 min, centrifuging to obtain 5-10 mL of supernatant, adding 0.01-0.5 mL of saturated lead acetate, and after liquid-liquid extraction, taking the aqueous phase.

[0014] Furthermore, in step 2), 1 μL-10mL of a 0.1-100 mM toluidine blue aqueous solution is added to 1 mL of the test solution.

[0015] Furthermore, the reaction conditions in step 2) are: reacting at 20-35° C. for 1-5 min.

[0016] Furthermore, the liquid-liquid extraction in step 2) is as follows: adding 0.5-2 mL of an organic solvent and shaking vigorously for 1-3 minutes; the organic solvent is one of ethyl acetate, ether, dichloromethane, and chloroform.

[0017] Further, the standard curve in step 3) is as follows: a series of perchlorate solutions with different standard concentrations are prepared, including a blank sample with a perchlorate concentration of 0, toluidine blue is added to form a reaction solution, after liquid-liquid extraction, the solution is allowed to stand, and the organic layer is taken for fluorescence detection, wherein the fluorescence intensity of the reaction solution of the perchlorate solution with a standard concentration of 0 is recorded as F0, and the fluorescence intensity of the reaction solution of the perchlorate solution with other standard concentrations is recorded as F, and △F represents the difference between F and F0, △F=F-F0; a perchlorate standard curve is obtained by plotting a series of different standard concentrations of the perchlorate solution as the horizontal coordinate and the difference in fluorescence intensity as the vertical coordinate.

[0018] Furthermore, the fluorescence detection system used in step 3) is as follows: a series of different standard concentrations of the perchlorate solution range from 0 to 0.005 mM, the excitation wavelength of the fluorescence method is 600 nm, and the emission wavelength is 670 nm.

[0019] The present invention has the advantages of simple operation, high sensitivity, short time consumption, low cost, good repeatability, etc. It can complete the visual detection and fluorescence detection of perchlorate in a short time, which is convenient for on-site real-time monitoring, and provides technical support for the qualitative determination of perchlorate in water and fresh tea leaves. The fluorescence detection limits in water and fresh tea leaves are 0.15 μM and 0.36 mg / kg, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a fluorescence characteristic diagram of different concentrations of perchlorate standard solutions after reaction in Example 1.

[0021] Figure 2 This is the standard curve for perchlorate fluorescence detection in Example 1.

[0022] Figure 3 This is the selective specificity of the color development reaction in Example 2 for perchlorate.

[0023] Figure 4 The relative fluorescence intensity change of perchlorate under 10 times the concentration of ions in Example 3. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and various embodiments.

[0025] Example 1: Color reaction of water and fresh tea leaf test solution

[0026] (1) Take water and fresh tea leaves for pretreatment to obtain the test solution. The water sample pretreatment is as follows: centrifuge river water and other water samples to obtain the supernatant; the tea leaf pretreatment is as follows: take 2g of fresh tea leaves, place them in 10-50mL of water, 0.1%-1% formic acid water or acetic acid aqueous solution, ultrasonically extract for 3-10min, centrifuge to obtain 5-10mL of supernatant, add 0.01-0.5mL of saturated lead acetate, and after liquid-liquid extraction, take the aqueous phase. If the fresh tea leaves are not purified, the pigments and substances in the extract will affect the color reaction, resulting in low detection sensitivity.

[0027] (2) Toluidine blue is added to the test solution obtained in step (1) as a color indicator to carry out a color reaction, wherein the amount of toluidine blue added is 1 μL-10 mL of a 0.1-100 mM toluidine blue aqueous solution added to 1 mL of the test solution, and the color reaction conditions are: reacting at 20-35° C. for 1-5 min; after the color reaction, liquid-liquid extraction is carried out, and the liquid-liquid extraction method is: adding 0.5-2 mL of an organic solvent, shaking vigorously for 1-3 min, and standing to separate layers, wherein the organic solvent includes one of ethyl acetate, ether, dichloromethane, and chloroform; the organic layer containing perchlorate appears blue, and the blank control organic layer appears red, thereby realizing rapid visual detection of perchlorate.

[0028] Example 2: Fluorescence detection of water and fresh tea leaf test solution

[0029] (1) Take water and fresh tea leaves for pretreatment to obtain the test solution. The water sample pretreatment is as follows: centrifuge river water and other water samples to obtain the supernatant; the tea leaf pretreatment is as follows: take 2g of fresh tea leaves, place them in 10-50mL of water, 0.1%-1% formic acid water or acetic acid aqueous solution, ultrasonically extract for 3-10min, centrifuge to obtain 5-10mL of supernatant, add 0.01-0.5mL of saturated lead acetate, and after liquid-liquid extraction, take the aqueous phase. If the fresh tea leaves are not purified, the pigments and substances in the extract will affect the color reaction, resulting in low detection sensitivity.

[0030] (2) Toluidine blue is added to the test solution obtained in step (1) as a fluorescent probe for fluorescence reaction. The amount of toluidine blue added is 1 μL-10 mL of a 0.1-100 mM toluidine blue aqueous solution added to 1 mL of the test solution. The color reaction conditions are: react at 20-35° C. for 1-5 min. After the color reaction, liquid-liquid extraction is performed. The liquid-liquid extraction method is: add 0.5-2 mL of an organic solvent, shake vigorously for 1-3 min, and let stand to separate layers.

[0031] (3) If the organic layer appears blue, it indicates that it contains perchlorate. The organic layer is subjected to fluorescence detection with an excitation wavelength of 600 nm. The fluorescence characteristic diagram after the fluorescence reaction of a series of perchlorate standard solutions with different concentrations (such as Figure 1), wherein the fluorescence intensity of the reaction solution of the perchlorate solution at a standard concentration of 0 is recorded as F0, and the fluorescence intensity of the reaction solution of the perchlorate solution at other standard concentrations is recorded as F, and △F represents the difference between F and F0. With the perchlorate concentration as the horizontal axis and the relative fluorescence intensity △F at the emission wavelength of 670nm as the vertical axis, a curve is drawn as follows Figure 2 The linear range of perchlorate in water is 0.0001-0.005 mmol / L, the corresponding standard curve is y=160206x-16.6, and the determination coefficient R 2 =0.9970; the standard curve of 0.0005-0.005 mmol / L perchlorate in fresh tea leaves is y=134.89 ln(x)+1040.4, and the determination coefficient R 2 =0.9926. The detection limit LOD = 3σ / k, σ is the standard deviation of 11 blank samples, k is the slope of the linear fitting curve, and the detection limits in water and fresh tea leaves are calculated to be 0.15μM and 0.36 mg / kg, respectively. Substitute the relative fluorescence intensity of the sample after reaction to calculate its content.

[0032] (4) Detection of samples: Take 1 mL of tap water (add perchlorate to make its concentration 0.001 mM) as an actual water environment sample, and add three groups in parallel to carry out the above fluorescence reaction. Measure the fluorescence intensity at 670 nm. The samples to be tested are measured three times in parallel. The tap water matrix external standard method is used for determination. The average recovery rate of perchlorate is 98.9%, and the relative standard deviation is 18.7%.

[0033] 2g of fresh tea leaves with one bud and two leaves were collected, and perchlorate was added to make the addition level reach 1 mg / kg. Three groups were paralleled, and after extraction and purification according to the pre-treatment process of fresh tea leaves, the above fluorescence reaction was carried out, and the fluorescence intensity at 670nm was measured. The samples to be tested were measured three times in parallel, and the fresh tea leaf matrix external standard method was used for determination. The average recovery rate of perchlorate reached 102.8%, and the relative standard deviation was 17.0%. The above results show that the fluorescence sensitization system of this embodiment is more reliable in determining perchlorate in water and fresh tea leaves, and meets the analysis requirements.

[0034] Example 3: Selective specificity and detection limit of color reaction

[0035] The same concentration of different anions (ClO4 - 、SO4 2- BrO3 - 、H2PO4 - 、HPO4 2- 、ClO2 - , Cl - 、F -, all sodium salt compounds, 0.005 mM), mix well, and let stand for 2 hours. Take 1 mL for color reaction, add 1 μL-10 mL of 0.1-100mM toluidine blue aqueous solution, react at 20-35℃ for 1-5 min, then add 0.5-2 mL of organic solvent (one of ethyl acetate, ether, dichloromethane, chloroform), shake vigorously for 1-3 min and let stand to separate (Figure 3). Figure 3 As shown, the organic layers of other anions are red, and only the organic layer of perchlorate is blue, which proves that the color reaction has good selectivity for perchlorate. Blank water and fresh tea leaves were taken, and different concentrations of perchlorate were added to perform the above color reaction, and the reaction detection limit was determined by colorimetry. The detection limit of the method of the present invention can reach 2 μM in water.

[0036] Example 4: Anti-interference ability of fluorescence detection

[0037] 0.1 mM perchlorate was added to deionized water, and then 10 times the concentration of different anions (no, SO4 2- BrO3 - 、H2PO4 - 、HPO4 2- 、ClO2 - , Cl - 、F - , all sodium salt compounds, 1 mM), mix well, and let stand for 2 hours. Take 1 mL for color development, add 1 μL-10 mL of 0.1-100 mM toluidine blue aqueous solution, react at 20-35°C for 1-5 min, then add 0.5-2 mL of organic solvent (one of ethyl acetate, ether, dichloromethane, chloroform), shake vigorously for 1-3 min, let stand and separate, take the organic layer for fluorescence analysis, under the excitation wavelength of 600nm, the relative peak intensity at the emission wavelength of 670nm remains unchanged, such as Figure 4 , indicating that the fluorescence detection method of perchlorate has strong anti-interference ability.

[0038] The above description is a preferred embodiment of the present invention, which is only used to explain the present invention and is not used to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for rapid visualization and fluorescence detection of perchlorate in water and fresh tea leaves, characterized in that: The following steps are involved: 1) Take water and fresh tea leaves for pretreatment to obtain the test solution; 2) Adding toluidine blue as a color indicator and a fluorescent probe to the test solution obtained in step 1) to react, after liquid-liquid extraction, the solution is allowed to stand, and the organic layer containing perchlorate appears blue, while the blank control organic layer appears red, thereby realizing rapid visual detection of perchlorate; 3) taking the organic layer for fluorescence detection to obtain the fluorescence intensity, substituting the value of the fluorescence intensity into the perchlorate standard curve to calculate the content of perchlorate in the sample to be tested; The standard curve is as follows: a series of perchlorate solutions with different standard concentrations are prepared, including a blank sample with a perchlorate concentration of 0, toluidine blue is added to form a reaction solution, after liquid-liquid extraction, the solution is allowed to stand, and the organic layer is taken for fluorescence detection, wherein the fluorescence intensity of the reaction solution of the perchlorate solution with a standard concentration of 0 is recorded as F0, and the fluorescence intensity of the reaction solution of the perchlorate solution with other standard concentrations is recorded as F, and △F represents the difference between F and F0, △F= F-F0; a perchlorate standard curve is obtained by plotting a series of different standard concentrations of the perchlorate solution as the horizontal coordinate and the difference in fluorescence intensity as the vertical coordinate.

2. A method for rapid visualization and fluorescence detection of perchlorate in water and fresh tea leaves as claimed in claim 1, characterized in that: The water pretreatment in step 1) is as follows: centrifuging the water sample and taking the supernatant.

3. The method for rapid visualization and fluorescence detection of perchlorate in water and fresh tea leaves as claimed in claim 1, characterized in that: The pretreatment of fresh tea leaves in step 1) is as follows: taking 2 g of fresh tea leaves, placing them in 10-50 mL of water or 0.1%-1% formic acid aqueous solution, ultrasonically extracting for 3-10 min, centrifuging to obtain 5-10 mL of supernatant, adding 0.01-0.5 mL of saturated lead acetate, and after liquid-liquid extraction, taking the aqueous phase.

4. The method for rapid visualization and fluorescence detection of perchlorate in water and fresh tea leaves according to claim 1, characterized in that: In step 2), 1 μL-10 mL of 0.1-100 mM toluidine blue aqueous solution is added to 1 mL of the solution to be tested.

5. The method for rapid visualization and fluorescence detection of perchlorate in water and fresh tea leaves as claimed in claim 1, characterized in that: The reaction conditions in step 2) are: reacting at 20-35° C. for 1-5 min.

6. A method for rapid visualization and fluorescence detection of perchlorate in water and fresh tea leaves as claimed in claim 1, characterized in that: The liquid-liquid extraction in step 2) is as follows: adding 0.5-2 mL of an organic solvent and shaking vigorously for 1-3 minutes; the organic solvent is one of ethyl acetate, ether, dichloromethane, and chloroform.

7. A method for rapid visualization and fluorescence detection of perchlorate in water and fresh tea leaves as claimed in claim 1, characterized in that: The fluorescence detection system used in step 3) is as follows: a series of different standard concentrations of the perchlorate solution range from 0 to 0.005 mM, the excitation wavelength of the fluorescence method is 600 nm, and the emission wavelength is 670 nm.

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