CN111233885A

CN111233885A - Fluorescent probe for detecting methanol and application thereof

Info

Publication number: CN111233885A
Application number: CN202010198447.XA
Authority: CN
Inventors: 高勇; 方明章; 邱建文; 钟春丽
Original assignee: Fujian Normal University
Current assignee: Fujian Normal University
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2020-06-05
Anticipated expiration: 2040-03-20
Also published as: CN111233885B

Abstract

The invention discloses a fluorescent probe for detecting methanol and application thereof, wherein the structural general formula of the fluorescent probe is shown as (I). The fluorescent probe for detecting methanol can selectively identify methanol, and has the characteristics of strong anti-interference performance and high sensitivity. The probe has no obvious response to other alcohol compounds, and can be used for measuring methanol in wines.

Description

Fluorescent probe for detecting methanol and application thereof

Technical Field

The invention relates to a fluorescent probe for detecting methanol, in particular to preparation of a methanol fluorescent probe and application of the methanol fluorescent probe in rapid detection of methanol.

Background

Methanol has high toxicity to human body, and the 5 th bulletin of 2004 of the national ministry of health indicates that "5-10 mL of methanol intake can cause poisoning, and if 30mL of methanol intake can cause death of human body". The metabolites of methanol in the body are formaldehyde and formic acid, which are more toxic than methanol, so that chronic poisoning can be caused by the very small intake of methanol. Methanol is colorless liquid with alcohol smell, is often used for manufacturing fake wine by lawless persons, and the methanol in the wine can exceed the standard due to the problems of untight control of the production process and the like of other wine brewing enterprises. Therefore, in the aspects of striking false wine cases, detecting the quality of wine commodities, diagnosing the methanol poisoning in clinic and the like, the methanol in the sample needs to be rapidly detected qualitatively and quantitatively.

Regarding the detection of the content of methanol in wine, the detection method given in the food safety standard GB 5009.266-2016 for the determination of methanol in food in China is gas chromatography. The current methods for detecting methanol reported in the literature include liquid chromatography and spectrophotometry. However, these methods require large-scale laboratory instruments, and the measurement process is complicated and time-consuming. Therefore, it is urgently required to develop a method for rapidly detecting methanol.

The fluorescence probe method has been developed in recent years, and an important analysis method for detecting a substance in a complicated environment has the advantages of rapidness, sensitivity, low cost and the like, and is widely applied to detection of various samples. Rhodamine is a dye containing a xanthene structure, has the advantages of high absorption coefficient, long excitation and emission wavelength, high fluorescence quantum yield, good light stability and the like, and is widely used for constructing various fluorescent probes as a parent of the fluorescent probes (chem. Rev.,2012, 112, 1910-.

Azo groups (-N = N-) are common structures in dye molecules, having both cis and trans configurations, and the ultrafast configuration change of the-N = N-bond causes fluorescence quenching of the fluorophore associated therewith (org. lett. 2014, 16, 15, 3946-. It has also been reported that the-N = N-bond of azo dyes can be reduced to an amino group by methanol under light conditions (Journal of the Society of Dyers and Colourists, 1982, 98, 334-. The invention designs a rhodamine azo derivative by utilizing the principle, and the ultra-fast configuration change of-N = N-bond of the compound leads the rhodamine group connected with the compound to generate fluorescence quenching; when the rhodamine azo derivative meets methanol under the illumination condition, the-N = N-bond is reduced and disconnected by the methanol, and the released rhodamine molecule is peach-red and emits strong fluorescence. The rhodamine azo derivative can be used for realizing the rapid detection of the methanol under the sunlight condition.

Disclosure of Invention

The invention aims to provide a methanol detection fluorescent probe and application thereof, the methanol fluorescent probe can realize fluorescence enhanced response to methanol, has strong anti-interference performance and high sensitivity, and has an application prospect of rapidly detecting a methanol sample on site.

The technical scheme adopted by the invention is as follows:

the structural general formula of the fluorescent probe for detecting methanol is shown as (I):

wherein: r = NO₂、Cl、Br、CN

（I）。

The preparation method of the fluorescent probe for detecting methanol comprises the following steps:

dissolving a certain amount of rhodamine compound in 2.5 mol/L dilute hydrochloric acid solution, fully stirring at room temperature, cooling the reaction solution to 0 ℃, and slowly adding a certain amount of NaNO₂Fully stirring for 20min, adding a certain amount of aniline compound, reacting for 20min, adjusting pH to neutral with 1mol/L NaOH solution, extracting the reaction solution with organic solvent for three times, combining organic phases, and reacting with anhydrous MgSO₄Fully drying, decompressing and steaming to remove the solvent, carrying out silica gel chromatography and column chromatography, and carrying out vacuum drying to obtain the product.

The rhodamine compound and NaNO described in the above method₂And the aniline compound in a molar ratio of 30:35: 32.

The organic solvent for extraction in the method is one of ethyl acetate, chloroform, dichloromethane and diethyl ether.

The silica gel chromatography eluent in the method is petroleum ether: ethyl acetate (V)_{Petroleum ether}：V_{Ethyl acetate}=20:1）。

The fluorescent probe for detecting the methanol can be used for quickly detecting a methanol sample.

The invention has the beneficial effects that: the fluorescent probe for detecting methanol can selectively identify methanol, and has the characteristics of strong anti-interference performance and high sensitivity. Due to the special response principle, the probe has no obvious response to other alcohol compounds, and can be used for measuring methanol in wine. The filter paper soaked by the probe solution can be used as methanol test paper to carry out qualitative detection on a methanol-containing sample.

Drawings

FIG. 1 is a selective fluorescence response spectrum of the methanol fluorescent probe 1 prepared in example 2 to a common solvent.

FIG. 2 shows fluorescence spectra and linear range (R) of methanol fluorescent probe 1 (20. mu.M) prepared in example 2 for methanol/water solutions (2-220. mu.M) of different concentrations²= 0.9788）。

Detailed Description

Example 1 Synthesis of rhodamine Compounds

Rhodamine compounds

3-diethylaminophenol (1.65 g, 10.00 mmol) was weighed into a 100 mL flask, 20 mL molecular sieve-dried toluene was added, and the mixture was stirred slowly at 60 ℃ until completely dissolved. Phthalic anhydride (1.50 g, ca. 10.13 mmol) was added under N₂The mixture was refluxed for 12h under protection. The pH was adjusted to 4 or less with dilute hydrochloric acid solution (ω = 20%) to obtain a purple-red precipitate. And (3) carrying out suction filtration on the product, fully washing the product by using distilled water, and drying the product to obtain 0.70 g of crude 4-diethylamino keto acid with the yield of 42.50%.

Weighing 1g (3.2 mmol) of crude 4-diethylaminoketoacid in a 100 mL flask, adding 10mL methanesulfonic acid, stirring at 50 deg.C to dissolve completely, adding 0.4 g (3.80 mmol) of m-aminophenol, and adding N₂Heating to 120 ℃ under protection and keeping the reaction for 10 h. After the reaction was complete, the reaction mixture was cooled to room temperature and then quenched with 0.1M Na₂CO₃Adjusting pH to 5 or less, extracting with ethyl acetate (3 × 20 mL), mixing organic phases, and MgSO₄Drying, suction filtering, reducingRemoving solvent by pressure evaporation, and purifying with silica gel column (V)_{Methylene dichloride}：V_Methanol=1: 1) to obtain 0.40 g of red solid, yield 40.35%.¹H NMR (400 MHz, CDCl₃) δ 8.03 (d, J = 7.1Hz, 1H), 7.57 (dtd, J = 19.6, 7.3, 1.1 Hz, 2H), 7.14 (d, J = 7.2 Hz, 1H),6.66 (d, J = 9.0 Hz, 1H), 6.57 (d, J = 8.6 Hz, 1H), 6.50 (d, J = 2.2 Hz, 1H),6.45 (d, J = 2.5 Hz, 1H), 6.39 (dd, J = 9.1, 2.5 Hz, 1H), 6.33 (dd, J = 8.6,2.2 Hz, 1H), 3.36 (q, J = 7.1 Hz, 4H), 1.16 (t, J = 7.1 Hz, 6H).¹³C NMR (101MHz, CDCl₃) δ 169.64, 153.87, 150.95, 150.48, 133.51, 129.58, 129.38, 129.21,125.99, 125.10, 112.47, 110.18, 109.05, 107.12, 100.53, 97.15, 44.60, 12.44。

Example 2 Synthesis of Probe 1

0.150 g (0.30 mmol) of the rhodamine compound obtained in example 1 was weighed in a 50 mL flask, 10mL of a 2.5 mol/L diluted hydrochloric acid solution was added, and the mixture was sufficiently stirred at room temperature. The flask was placed in a solid ice-water mixture with sodium chloride and stirred for 5 min. Weighing NaNO₂Slowly adding solid 0.024g (0.35 mmol) into the reaction solution, stirring thoroughly for 20min, adding p-nitroaniline 0.044g (0.32 mmol), reacting for 20min, and adjusting pH to neutral with 1mol/L NaOH solution. The reaction solution was further extracted thoroughly with ethyl acetate (3X 30 mL), and the organic phases were combined and extracted with anhydrous MgSO₄Drying thoroughly, evaporating the solvent under reduced pressure, and purifying with silica gel column chromatography (V)_{Petroleum ether}：V_{Ethyl acetate}=20: 1), vacuum drying to obtain 10.038 g of a probe, and the yield is 23.50%.¹HNMR (400 MHz, CDCl₃) δ 8.26 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 7.4 Hz, 1H),7.68 (dd, J = 13.2, 7.2 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.41 (s, 1H), 7.24(d, J = 7.6 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.62(d, J = 8.6 Hz, 1H), 6.47 (s, 1H), 6.41 (d, J = 8.7 Hz, 1H), 3.39 (dd, J =13.8, 6.9 Hz, 4H), 1.21 (t, J = 6.9 Hz, 6H).¹³C NMR (101 MHz, CDCl₃) δ169.56,153.04, 152.54, 142.34, 134.93, 132.57, 129.70, 129.20, 127.95, 125.57,124.99, 124.06, 117.95, 116.37, 115.91, 108.42, 100.97, 100.00, 99.90, 97.80,44.57, 41.61,37.12, 32.77, 29.70, 24.80, 21.06, 18.05, 14.12, 12.50, 11.34.MS (ESI): [M+H⁺]anal. calcd for: C₃₀H₂₅N₅O₅:536.1928 ; found: 536.1907。

EXAMPLE 3 Synthesis of Probe 2

20.056 g of probe was synthesized in the same manner as in example 2, and the yield was 43.33%.¹H NMR (400 MHz, CDCl₃) δ8.05 (d, J = 7.4 Hz, 1H), 7.66 (dt, J = 23.3, 7.4 Hz, 2H), 7.45 (d, J = 8.6Hz, 2H), 7.38 (d, J = 8.7 Hz, 2H), 7.30 (s, 1H), 7.24 (d, J = 7.4 Hz, 1H),6.94 (d, J = 8.5 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.60 (d, J = 8.9 Hz, 1H),6.49 (s, 1H), 6.39 (d, J = 9.1 Hz, 1H), 3.39 (q, J = 7.0 Hz, 4H), 1.20 (t, J= 7.0 Hz, 6H).¹³C NMR (101 MHz, CDCl₃) δ 169.71, 153.11, 152.90, 152.73,149.65, 145.18, 144.83, 134.86, 131.92, 129.56, 129.35, 128.93, 127.24,124.96, 124.06, 120.72, 115.32, 112.04, 108.47, 104.97, 103.91, 97.55, 84.24,44.52, 37.12, 31.95, 29.73, 22.72, 14.16, 12.53. MS (ESI): [M+H⁺]anal. calcdfor: C₃₀H₂₅ClN₄O₃:525.1687 ; found: 525.1692。

EXAMPLE 4 Synthesis of Probe 3

30.081 g of probe was synthesized according to the method of example 2, and the yield was 54.26%.¹H NMR (400 MHz, CDCl₃) δ8.06 (d, J = 7.6 Hz, 1H), 7.66 (dt, J = 22.5, 7.3 Hz, 2H), 7.52 (d, J = 8.2Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.30 (s, 1H), 7.24 (d, J = 7.5 Hz, 1H),6.95 (d, J = 8.5 Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 6.61 (d, J = 8.9 Hz, 1H),6.49 (s, 1H), 6.39 (d, J = 9.0 Hz, 1H), 3.39 (q, J = 6.9 Hz, 4H), 1.20 (t, J= 6.9 Hz, 6H).¹³C NMR (101 MHz, CDCl₃) δ 169.71, 153.09, 152.91, 152.72,149.68, 145.42, 145.07, 134.84, 132.29, 129.55, 129.30, 128.91, 127.25,124.96, 124.06, 120.88, 119.65, 115.49, 112.26, 108.52, 105.52, 104.14,97.61, 44.52, 31.94, 29.71, 29.37, 22.71, 14.14, 12.53. MS (ESI): [M+H⁺]anal. calcd for: C₃₀H₂₅BrN₄O₃:569.1128 ; found: 569.1187。

EXAMPLE 5 Synthesis of Probe 4

40.070 g of probe was synthesized according to the method of example 2, and the yield was 46.67%.¹H NMR (400 MHz, CDCl₃) δ8.05 (d, J = 7.5 Hz, 1H), 7.66 (dt, J = 23.1, 7.4 Hz, 2H), 7.41 (d, J = 7.8Hz, 2H), 7.30 (s, 1H), 7.23 (t, J = 7.9 Hz, 3H), 6.94 (d, J = 8.5 Hz, 1H),6.76 (d, J = 8.5 Hz, 1H), 6.61 (d, J = 8.9 Hz, 1H), 6.50 (s, 1H), 6.39 (d, J= 8.9 Hz, 1H), 3.39 (q, J = 6.9 Hz, 4H), 2.38 (d, J = 10.3 Hz, 3H), 1.20 (t,J = 7.0 Hz, 6H).¹³C NMR (101 MHz, CDCl₃) δ 169.70, 153.19, 152.94, 152.73,149.64, 145.30, 144.20, 136.60, 134.79, 129.84, 129.49, 129.26, 128.91,127.27, 124.92, 124.07, 119.40, 114.89, 111.97, 108.44, 105.12, 103.72,97.63, 84.29, 44.52, 31.94, 29.71, 22.71, 21.11, 14.14, 12.53. MS (ESI): [M+H⁺]anal. calcd for: C₃₁H₂₈N₄O₃:505.2234; found: 505.2217。

Example 6 Probe Performance testing

The performance test was performed by taking the probe 1 prepared in example 2 as an example.

1) Fluorescence spectrometry of the probe 1 prepared in example 2 in various solvents. A certain amount of stock solution (1 mM) of probe 1 was added to each of the different solvents so that the final concentration of probe 1 was 20. mu.M. The fluorescence spectrum of the obtained sample was measured, and the results are shown in FIG. 1 (excitation light wavelength: 480 nm). As can be seen from FIG. 1, probe 1 has a good fluorescence response to methanol, a weak fluorescence response to ethanol, and no fluorescence response to other common solvents.

2) Fluorescent response of probe 1 to various concentrations of aqueous methanol solution. A certain amount of the stock solution of probe 1 (1 mM) was added to aqueous solutions of different methanol concentrations (2-220. mu.M), respectively, so that the final concentration of probe 1 was 20. mu.M. The fluorescence spectrum of the obtained sample was measured, and the result is shown in FIG. 2 (excitation light wavelength: 480 nm). As can be seen from FIG. 2, the fluorescence intensity of the probe gradually increased as the methanol concentration increased. The fluorescence intensity is linear with methanol concentration in the range of 2-220. mu.M (R)²= 0.9788), the probe can quantitatively detect methanol.

3) And detecting the methanol by using the probe 1 test paper. And immersing the filter paper strip into the stock solution (1 mM) of the probe 1 for 10 seconds, and naturally airing the filter paper strip to obtain the methanol test paper. And (3) dropwise adding an ethanol solution with the methanol content of 0.1% onto the test paper, and standing for 1 min to obtain a peach-red test paper. There was no apparent phenomenon when the test paper was dropped with pure ethanol. The experiments show that the test paper can conveniently detect trace methanol in ethanol.

Claims

1. A fluorescent probe for detecting methanol is shown in a structural general formula (I):

wherein: r = NO₂、Cl、Br、CN

（I）。

2. The method for preparing a fluorescent probe for detecting methanol according to claim 1, comprising the steps of:

3. The method for preparing a fluorescent probe for detecting methanol as claimed in claim 2, wherein the rhodamine compound and NaNO are used₂And the aniline compound in a molar ratio of 30:35: 32.

4. The method for preparing a fluorescent probe for detecting methanol as claimed in claim 2, wherein the organic solvent for extraction is one of ethyl acetate, chloroform, dichloromethane and diethyl ether.

5. The method for preparing a fluorescent probe for detecting methanol according to claim 2, wherein the silica gel chromatography eluent is petroleum ether: acetic acid ethyl ester, V thereof_{Petroleum ether}：V_{Ethyl acetate}=20:1。

6. A fluorescent probe for detecting methanol according to claim 1, which is used for rapid detection of a methanol sample.