CN102735662A - High sensitivity and high selectivity fluorescence emission spectrum analysis method for zinc ions - Google Patents

Abstract

The invention establishes a fluorescence spectroscopic analysis method for detecting zinc ions by using the compound bis[(1-hydroxyl 2-naphthyl)-methine]hydrazone as a probe. The method is strictly controlled in the DMF/H ₂ O mixture containing 10% H ₂ O, adjusted to pH~8 with Tris-HCl buffer solution, and the compound bis[(1-hydroxyl, 2-naphthyl)-methine Base] hydrazone as a fluorescent chromogenic agent for detecting Zn ²⁺ , and the fluorescence intensity of the solution was measured to determine the content of Zn ²⁺ in the solution. Specific medium conditions are designed to make the compound a fluorescence-enhanced probe for detecting zinc ions, and implement highly sensitive and selective recognition detection of zinc ions in different samples.

Description

A highly sensitive and highly selective fluorescence emission spectrometric analysis method for zinc ions

technical field

The invention discloses a highly sensitive and highly selective fluorescence emission spectroscopic analysis method for zinc ions, which belongs to the field of analytical chemistry.

Background technology: In the field of molecular recognition, fluorescent probes with molecular device properties selectively bond with target substances to change the parameters of fluorescence enhancement or quenching, fluorescence polarization, wavelength and peak shape, and fluorescence lifetime before and after binding. The role of the microscopic field is expressed through optical information, so as to realize in-situ real-time detection at the molecular level, and achieve effective identification of metal ions, organic molecules, biomolecules, etc. The so-called "fluorescent probe" technology is used to combine probe molecules with non-fluorescent or weakly fluorescent substances to form fluorescent complexes, supramolecules or aggregates in covalent or other forms. Due to the high sensitivity and high selectivity of fluorescence analysis, real-time in situ detection, simple equipment, and can provide rich spectral information, various ion detection and protein molecular labeling in analytical chemistry, biochemistry, environmental science, pharmaceuticals and other fields , DNA and cell detection, immune analysis and other aspects play an important role.

Organic fluorescent probes are mainly represented by fluorescein, rhodamine, acridine, and coumarin, which have high fluorescence quantum yield, long excitation wavelength, and wide application range. Among them, a large number of fluorescence-enhancing or quenching rhodamine probes capable of detecting Hg ²⁺ , Cu ²⁺ , Pb ²⁺ , Cr ³⁺ , and Fe ³⁺ ions have been reported. Aromatic compounds are often used as fluorescent signal generating groups of organic fluorescent probes. The fused-ring aromatic hydrocarbons mainly represented by naphthalene, anthracene, pyrene, quinoline, cyanines, pyrromethenes, etc. have strong and stable fluorescence and large Stokes shift. In the field of fluorescent probes, as model fluorophores, the The constructed fluorescent probe is a kind of sensor with excellent performance. For example, fluorescent probes with structures such as calixarene and tripod with naphthalene as the fluorescent group show recognition of Zn ²⁺ , Hg ²⁺ , Cu ²⁺ ions, and quinazoline as Co ²⁺ ion fluorescence quenching The study of type probes has also been reported.

Zinc is one of the essential trace elements for the human body and is widely distributed in the cells and body fluids of the human body. Among transition metal ions, its content is second only to iron ions, and is closely related to human health. It is necessary to establish a detection method for trace Zn ²⁺ in the fields of plants, food, medicine and clinical.

It has a wide range of application fields for the highly sensitive and selective detection and identification of trace metal ions in the fields of biological systems, physiological processes, environment and food safety. It is of great significance to find fluorescent probes that are different from traditional organic fluorescent dye molecules, make sample processing simple, fast measurement method, low test cost, and can simultaneously detect multiple metal ions separately, with superior performance. Trace zinc detection methods include atomic absorption spectrometry, atomic emission spectrometry, inductively coupled plasma, mass spectrometry, ultraviolet-visible spectrophotometry, fluorescence and other methods. Fluorescence spectroscopy has more application value due to its simple operation and no need for expensive instruments and equipment. However, due to the poor water solubility of most chromogenic reagents, complex pretreatments such as extraction and separation are required before they can be used for detection. The key problem is that the sensitivity and selectivity of detection cannot meet the increasing demand. Fluorescence signal has incomparable advantages in sensitivity. At present, there are few reports on the use of fluorescence methods to detect zinc ion content. It is extremely challenging to find new fluorescent probes with sufficient selectivity and sensitivity for Zn ²⁺ and application value. The inventors of the present invention found that the compound bis[(1-hydroxyl, 2-naphthyl)-methine]hydrazone, in a mixed solvent of DMF/H ₂ O (V/V, 9/1), On the fluorescence spectrophotometer, the excitation wavelength is 438nm, and the fluorescence emission wavelength is 516nm. When Zn ²⁺ exists, the fluorescence of the probe compound can be enhanced. Within a certain concentration range of Zn ²⁺ , the concentration of Zn ²⁺ and Fluorescence emission intensity is proportional, common coexisting metal ions do not interfere with the determination, and has high sensitivity and high selectivity, laying the foundation for a highly sensitive and highly selective fluorescence emission spectroscopic analysis method of zinc ions in the present invention.

Summary of the invention: The object of the present invention is to use the fluorescence spectrophotometry method to quantitatively detect the zinc ion content of the sample, and to create a new fluorescence emission spectrum quantitative analysis method with sufficient selectivity and sensitivity to the Zn ²⁺ concentration.

A highly sensitive and highly selective fluorescence emission spectrum analysis method for zinc ions of the present invention is characterized in that the compound di[(1-hydroxyl, 2-naphthyl)-methine]hydrazone is used as a probe, and the fluorescence emission spectrum method is adopted Quantitatively measure the concentration of zinc ion, the chemical structural formula of compound two [(1-hydroxyl, 2-naphthyl)-methine] hydrazone is

The molecular formula is C ₂₂ H ₁₆ N ₂ O ₂ , and the English name is Bis(1-hydroxy-2-naphthyl)-methylene hydrazone. The analysis method adopts the standard curve method: take several 10.0ml volumetric flasks, and add 1ml of the probe compound solution with a concentration of 0.10 mmol·L ^-1 and 1ml of Tris-HCl buffer solution, pH~8, into each volumetric flask. Then add different concentrations of Zn ²⁺ standard solutions into each volumetric flask, add sample solution to one of the volumetric flasks, and finally use a mixture of DMF and H ₂ O to make up to 10.0ml, shake well, and place at a constant temperature of 25°C for half an hour , on a fluorescence spectrophotometer, use 438nm as the excitation wavelength and 516nm as the emission wavelength to measure the fluorescence intensity, draw the calibration curve of the fluorescence intensity to the standard Zn2 ⁺ concentration, and use the calibration curve to obtain the sample solution according to the fluorescence emission intensity of the sample solution. The concentration of Zn ²⁺ in the solution is used to calculate the content of the sample. The concentration of Zn ²⁺ in the standard solution is in the range of 8.00×10 ^-6 ~2.00×10 ^-4 mol·L ^-1 . Zn ²⁺ increases the fluorescence intensity of the probe linearly relationship; other coexisting ions including alkali metals, alkaline earth metals, transition metals, heavy metals, rare earth metals, and cobalt ions have less than 5% impact on the fluorescence intensity of zinc ion detection when the concentration is the same as that of Zn ²⁺ . The chemical name of DMF is N,N-dimethylformamide, the molecular formula is HCON(CH ₃ ) ₂ , and it is a common organic solvent. The volume ratio of the mixed solution of DMF and H ₂ O is 9:1, and the probe compound solution referred to is the DMF solution of two [(1-hydroxyl, 2-naphthyl)-methine] hydrazone; the referred Tris is Trishydroxymethylaminomethane. The present invention is also applicable to the standard addition method: get 6 10.0ml volumetric flasks, add 1ml of sample solution, 1ml of probe compound solution with a concentration of 0.10 mmol L ^-1 , 1ml of Tris-HCl buffer solution in each volumetric flask, pH~8. Then do not add Zn ²⁺ standard solution to the first volumetric flask, add Zn ²⁺ standard solution with the same concentration and different volumes in turn in the remaining volumetric flasks, and finally use the mixed solution of DMF and H ₂ O to adjust the volume to 10.0ml, Shake well, and place at a constant temperature of 25°C for half an hour. On a fluorescence spectrophotometer, use 438nm as the excitation wavelength and 516nm as the emission wavelength to measure the fluorescence intensity in turn, and draw the calibration curve of the fluorescence intensity versus the volume of the Zn ²⁺ standard solution added. According to the straight line Extrapolating to the point where the ordinate is zero and the abscissa intersects, the concentration of Zn ²⁺ in the sample solution can be obtained, and the sample content can be calculated.

Above-mentioned is that the preparation method of various reagents is:

(1) Preparation method of the probe compound solution: weigh 3.4 mg of bis[(1-hydroxy, 2-naphthyl)-methine]hydrazone, dissolve it in DMF, and prepare a 100 mL solution with a concentration of 0.100 mmol L ^-1 ;

(2) Zn ²⁺ standard solution: Weigh 27.2 mg of high-grade pure ZnCl ₂ , dissolve it in DMF, and prepare a 100mL solution. The concentration of Zn ²⁺ is 2.00×10 ^-3 mol·L ^-1 ; Diluted to the appropriate concentration;

(3) Preparation of Tris-HCl buffer solution: Weigh 0.242 g Tris and dissolve it in DMF to prepare a 500 mL solution with a concentration of 0.004 mol L ^-1 , pipette a certain volume of Tris and mix it with an appropriate amount of HCl, and adjust the pH value to 8.

(4) Preparation of other coexisting ionic solutions: Dissolve nitrates or hydrochlorides of various metals with superior grades in DMF, and prepare a DMF solution with a concentration of 2.00×10 ^-3 mol·L ^-1 .

When the above-mentioned analytical method of the present invention takes volume change, various solution additions will make corresponding change in proportion.

When the present invention measures zinc ions, the coexisting ions Na ⁺ , K ⁺ , Rb ⁺ , Mg ²⁺ , Ca ²⁺ , Sr ²⁺ , Ba ²⁺ , Pb ²⁺ , Bi ²⁺ , Mn ²⁺ , Ni ²⁺ , Cu ²⁺ , Cd ²⁺ , Hg ²⁺ , Al ³⁺ , Fe ³⁺ , Sc ³⁺ , Ti ³⁺ , Cr ³⁺ and Co ²⁺ , when the concentration is comparable to Zn ²⁺ , detect Zn ²⁺ The relative deviations of the fluorescence intensity effects are all within 5%. Neither interfered with the measurement.

The reagents used in the method of the present invention are analytically pure reagents, and the water used is twice distilled water.

Instrument used in the present invention is a fluorescence spectrophotometer, and the model is a Cary Eclipse fluorescence spectrophotometer, produced by U.S. VARIAN Company.

The method of the invention has the advantages of large Stokes shift, high selectivity, extremely low detection limit, no need for separation, fast response time, close to neutral test conditions and the like. It can be used to detect Zn ²⁺ content in environmental water monitoring (polluted water), drug analysis (zinc adjuvant therapeutic agent), food inspection (zinc-containing beverages) and other fields.

The compound bis[(1-hydroxy2-naphthyl)-methine]hydrazone molecule exhibits weak fluorescence in a benign solvent (DMF/H ₂ O, 9/1), and can form with metal zinc ions under certain conditions Determine the coordination ratio of the complex. The key technology in the patent of this invention is to control the ratio of different DMF/H ₂ O mixed solvents and a certain pH value, so that the compound bis[(1-hydroxyl 2-naphthyl)-methine]hydrazone can be used to detect Zn ²⁺ Fluorescence-enhanced probes for ions. The method of operation and control is simple and the performance is unique, which is the practical application of the organic molecule as a fluorescent probe.

Description of drawings :

Figure 1 Compound bis[(1-hydroxy2-naphthyl)-methine]hydrazone (10 μM, DMF/H ₂ O, 10% H ₂ O, Tris-HCl, pH~8) detection and recognition of zinc ion (200 μM), other metal ions (200μM): Na ⁺ , K ⁺ , Rb ⁺ , Mg ²⁺ , Ca ²⁺ , Sr ²⁺ , Ba ²⁺ , Pb ²⁺ , Bi ²⁺ , Mn ²⁺ , Ni ²⁺ , Cu ²⁺ , Cd ²⁺ , Hg ²⁺ , Al ³⁺ , Fe ³⁺ , Sc ³⁺ , Ti ³⁺ , Cr ³⁺ and Co ²⁺ do not interfere with the determination within the error range, from the figure It can be seen that the pH of the solution is controlled at about 8, the volume ratio of DMF/H ₂ O mixed solvent is 1:9, and the fluorescence intensity of the probe compound is very weak. Under this condition, adding Zn ²⁺ ions can make the probe compound solution The emission of strong fluorescence at the wavelength, while other experimental metal ions including Co ²⁺ ions cannot significantly enhance the fluorescence.

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Fig. 2 Fluorescence intensity of coexisting metal ions when the compound bis[(1-hydroxyl 2-naphthyl)-methine]hydrazone is used as a probe to detect Zn ²⁺ . White: probe; or probe + metal ion; black: probe + Zn ²⁺ + metal ion. It can be seen from the figure that the fluorescence emission of the probe compound is very weak in the medium of DMF/H ₂ O (V/V, 1:9) at pH 8 (the white bar on the left in the figure). The fluorescence is still very weak and has little effect on its intensity (from 3 to 20 on the left in the figure, the white bars corresponding to metal ions). After adding Zn ²⁺ , the fluorescence of the probe is significantly enhanced (the 2 white bars on the left in the figure). The effects of adding other metal ions to the needle and the solution with cobalt ions on the fluorescence enhancement of the probe by zinc ions are within the error range (black bars in the figure).

Fig. 3 Calibration curve for detection of Zn ²⁺ . The ordinate represents the fluorescence intensity, and the abscissa represents the concentration of the added Zn ²⁺ standard solution.

Fig. 4 Fluorescence spectra of Zn ²⁺ detected in lake water samples. The spectrograms measured by adding different amounts of Zn ²⁺ standard solutions to the sample solution in turn according to the standard addition method.

Fig. 5 Fluorescence spectra for detection of Zn ²⁺ content in zinc gluconate samples. The spectrograms of the two samples were measured in parallel according to the calibration curve method.

Specific implementation method:

Example 1: Detection of Zn ²⁺ content in polluted lake water.

1. Pretreatment of lake water samples: Take 2.0 L of polluted lake water samples, boil them for 10 minutes, cool them down, let them stand for 12 hours, filter them, and test the pH value of the water samples. Concentrate to 10ml, cool for later use.

2. Detection: pipette 1mL of the treated lake water sample into a 10mL volumetric flask, add 1 mL of probe compound (10 μM, DMF), 1 mL of Tris-HCl, add 0, 1, 2 mL of Zn ²⁺ ( 10 μM), dilute to volume with aqueous 10% DMF/H ₂ O mixture, and place at a constant temperature of 25°C for half an hour. Measured 3 times in parallel.

3. The measured spectrogram is shown in Figure 4, and the results calculated according to the standard addition method are shown in Table 1.

Table 1 Detection of Zn ²⁺ in lake water samples

Embodiment 2: Determination of zinc content in commercially available zinc gluconate.

Detect the content of zinc in a certain brand of zinc gluconate lozenges:

1. Pretreatment: Take a piece of zinc gluconate 32mg (0.32mg as zinc) and grind it into powder, dissolve it in a 100mL volumetric flask. Vibrate in ultrasonic for 30 minutes, then stand still for 2 hours and take the supernatant.

2. Detection process: Add 1 mL of probe compound (10 μM, DMF), add 1 mL of Tris-HCl (pH = 8), pipette 1 mL of the above prepared sample into a 10 mL volumetric flask, and use 10% DMF/H ₂ O mixed solution to volume. Place it at a constant temperature of 25°C for half an hour, and measure its fluorescence intensity at 514nm. Parallel measurement was performed 2 times.

3. See attached drawing 5 for the measured spectrogram, and calculate the following results according to the calibration curve obtained in the experiment, see Table 3.

Claims (7)

1. a kind of highly sensitive, highly selective fluorescence emission spectrometry analysis method of zinc ion, it is characterized in that taking compound two [(1-hydroxyl, 2-naphthyl)-methine] hydrazone as probe, adopts fluorescence emission spectrometry Quantitative determination of zinc ion concentration, the chemical structural formula of the compound bis[(1-hydroxyl, 2-naphthyl)-methine]hydrazone is

The molecular formula is C ₂₂ H ₁₆ N ₂ O ₂ , the English name is Bis(1-hydroxy-2-naphthyl)-methylene hydrazone, and the analysis method adopts the standard calibration curve method: take several 10.0ml volumetric flasks, and add 1ml of probe compound solution with a concentration of 0.10 mmol L ^- 1, 1ml of Tris-HCl buffer solution, pH~8, and then add different concentrations of Zn ²⁺ standard solutions to each volumetric flask, and add sample solution to one of the volumetric flasks , and finally use a mixture of DMF and H ₂ O to make up to 10.0ml, shake well, and place at a constant temperature of 25°C for half an hour. On a fluorescence spectrophotometer, use 438nm as the excitation wavelength and 516nm as the emission wavelength to measure the fluorescence intensity sequentially, and draw Get the calibration curve of fluorescence intensity to standard Zn ²⁺ concentration, according to the fluorescence emission intensity of the sample solution, use the calibration curve to obtain the concentration of Zn ²⁺ in the sample solution, calculate the sample content, the Zn ²⁺ concentration in the standard solution is 8.00×10 In the range of ^-6 ~2.00×10 ^-4 mol·L ^-1 , Zn ²⁺ increases the fluorescence intensity of the probe in a linear relationship; other coexisting ions include alkali metals, alkaline earth metals, transition metals, heavy metals, rare earth metals and cobalt ions in the When the concentration is the same as that of Zn ²⁺ , the relative deviation of the influence on the fluorescence intensity of Zn ²⁺ detection is within 5%. The chemical name of DMF is N, N-dimethylformamide, and the molecular formula is HCON(CH ₃ ) ₂ , It is a conventional organic solvent, the volume ratio of DMF and H ₂ O mixture is 9:1, and the probe compound solution referred to is the DMF solution of bis[(1-hydroxyl, 2-naphthyl)-methine]hydrazone ; The Tris referred to is trihydroxymethylaminomethane. 2. the high sensitivity of a kind of zinc ion according to claim 1, the highly selective fluorescence emission spectrometry analysis method, it is characterized in that analysis method is applicable to standard addition method equally: get 6 10.0ml volumetric flasks, in each volumetric flask Add 1ml of sample solution, 1ml of probe compound solution with a concentration of 0.10 mmol·L ^-1 , 1ml of Tris-HCl buffer solution, pH

8. Then do not add Zn ²⁺ standard solution to the first volumetric flask, add Zn ²⁺ standard solution with the same concentration and different volumes in turn to the remaining volumetric flasks, and finally use the mixed solution of DMF and H ₂ O to adjust the volume to 10.0 ml, shake well, place at a constant temperature of 25°C for half an hour, measure the fluorescence intensity sequentially on a fluorescence spectrophotometer with 438nm as the excitation wavelength and 516nm as the emission wavelength, draw the calibration curve of the fluorescence intensity versus the volume of the Zn ²⁺ standard solution added, According to the straight line extrapolation to the point where the ordinate is zero and the abscissa intersects, the concentration of Zn ²⁺ in the sample solution can be obtained and the sample content can be calculated. 3. according to claim 1 and 2 described a kind of highly sensitive, highly selective fluorescence emission spectroscopic analysis method of zinc ion, it is characterized in that the preparation method of various reagents is: (1) Preparation method of the probe compound solution: weigh 3.4 mg of bis[(1-hydroxy, 2-naphthyl)-methine]hydrazone, dissolve it in DMF, and prepare a 100 mL solution with a concentration of 0.100 mmol L ^-1 ; (2) Zn ²⁺ standard solution: Weigh 27.2 mg of high-grade pure ZnCl ₂ , dissolve it in DMF, and prepare a 100mL solution. The concentration of Zn ²⁺ is 2.00×10 ^-3 mol·L ^-1 ; Diluted to the appropriate concentration; (3) Preparation of Tris-HCl buffer solution: Weigh 0.242 g Tris and dissolve it in DMF to prepare a 500 mL solution with a concentration of 0.004 mol L ^-1 , pipette a certain volume of Tris and mix it with an appropriate amount of HCl, and adjust the pH value to 8; (4) Preparation of other coexisting ionic solutions: Dissolve nitrates or hydrochlorides of various metals with superior grades in DMF, and prepare a DMF solution with a concentration of 2.00×10 ^-3 mol·L ^-1 . 4. according to claim 1 and 2 described a kind of highly sensitive, highly selective fluorescence emission spectrometry analysis method of a kind of zinc ion, it is characterized in that when volumetric flask volume changes, various solution add-ons will make corresponding change in proportion. 5. The highly sensitive and highly selective fluorescence emission spectrometric analysis method of a kind of zinc ion according to claim 1 or 2, it is characterized in that other coexisting ions include: Na ^{+ ,} K ⁺ , Rb ⁺ , Mg when measuring zinc ion ⁺ , Ca ²⁺ , Sr ²⁺ , Ba ²⁺ , Pb ²⁺ , Bi ²⁺ , Mn ²⁺ , Ni ²⁺ , Cu ²⁺ , Cd ²⁺ , Hg ²⁺ , Al ³⁺ , Fe ³⁺ , Sc ³⁺ , Ti ³⁺ , Cr ³⁺ and Co ²⁺ do not interfere with the determination when the concentration is equivalent to Zn ²⁺ . 6. according to claim 1 and 2 described a kind of highly sensitive, highly selective fluorescence emission spectrometry analysis method of a kind of zinc ion, it is characterized in that used reagent is analytical pure reagent, and used water is twice distilled water. 7. the highly sensitive, highly selective fluorescence emission spectrometry analysis method of a kind of zinc ion according to claim 1 or 2, it is characterized in that the instrument used for analysis is a fluorescence spectrophotometer.

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