CN111233885A - A Fluorescent Probe for Detecting Methanol and Its Application - Google Patents

Abstract

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

Description

A Fluorescent Probe for Detecting Methanol and Its Application

technical field

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

Background technique

Methanol is highly toxic to the human body, and the Ministry of Health of the People's Republic of China stated in the 2004 Announcement No. 5 that "ingestion of 5-10 mL of methanol can cause poisoning, and if ingested 30 mL, it can cause death." The metabolites of methanol in the body are formaldehyde and formic acid, which are more toxic than methanol, so the human body can also cause chronic poisoning by ingesting a very small amount of methanol. Methanol, as a colorless liquid with an alcoholic odor, is often used by some criminals to make fake wine. In addition, some brewing companies have problems such as lax production process control, which will also lead to excessive methanol in wine. Therefore, it is necessary to quickly conduct qualitative and quantitative detection of methanol in samples in the fight against counterfeit wine cases, quality testing of alcoholic products, and clinical diagnosis of methanol poisoning.

Regarding the detection of methanol content in wine, the detection method given in my country's food safety standard "GB 5009.266-2016 Determination of Methanol in Food" is gas chromatography. At present, the methanol detection methods reported in the literature include liquid chromatography, spectrophotometry and so on. However, these methods all require large-scale experimental instruments, and the measurement process is complicated and time-consuming. Therefore, it is urgent to develop a method for rapid detection of methanol.

Fluorescent probe method has been developed in recent years and is an important analytical method for the detection of substances in complex environments. It has the advantages of rapidity, sensitivity and low cost, and is widely used in the detection of various samples. Rhodamine is a dye containing xanthene structure, which has the advantages of high absorption coefficient, long excitation and emission wavelength, high fluorescence quantum yield and good photostability. for the construction of various fluorescent probes (Chem. Rev., 2012, 112, 1910-1956).

The azo group (-N=N-) is a common structure in dye molecules, with both cis and trans configurations, and the ultra-rapid configuration change of the -N=N- bond can make the fluorescence associated with it The group undergoes fluorescence quenching (Org. Lett. 2014, 16, 15, 3946-3949). It has also been reported that the -N=N- bonds of azo dyes can be reduced to amino groups by methanol under light conditions (Journal of the Society of Dyers and Colourists, 1982, 98, 334-340). The present invention utilizes the above principles to design a rhodamine azo derivative. The ultra-rapid configuration change of the -N=N- bond of the compound causes fluorescence quenching of the associated rhodamine group; when the rhodamine azo derivative is When the derivative encounters methanol under light conditions, the -N=N- bond is cut off by methanol reduction, and the released rhodamine molecule is pink and emits strong fluorescence. The present invention utilizes the above-mentioned rhodamine azo derivatives to realize rapid detection of methanol under sunlight conditions.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a fluorescent probe for methanol detection and its application. The methanol fluorescent probe can realize enhanced fluorescence response to methanol, has strong anti-interference and high sensitivity, and has application prospects for rapid detection of methanol samples on site. .

The technical scheme adopted in the present invention is:

The general structural formula of a fluorescent probe for methanol detection is shown in (I):

Where: R = NO ₂ , Cl, Br, CN

(I).

The preparation method of the fluorescent probe for methanol detection is as follows:

A certain amount of rhodamine compound was dissolved in a 2.5 mol/L dilute hydrochloric acid solution, fully stirred at room temperature, the reaction solution was cooled to 0 °C, a certain amount of NaNO ₂ was slowly added, and after fully stirring for 20 min, a certain amount of The aniline compounds were reacted for another 20 min, and then adjusted to neutral pH with 1 mol/L NaOH solution, the reaction solution was extracted three times with organic solvent, the organic phases were combined, fully dried with anhydrous MgSO ₄ , and the solvent was evaporated under reduced pressure. Silica gel chromatography was passed through the column, and the product was obtained by vacuum drying.

The molar ratio of the rhodamine compound, NaNO ₂ and the aniline compound described in the above method is 30:35:32.

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

The silica gel chromatography eluent described in the above method is petroleum ether: ethyl acetate (V _{petroleum ether} : V _{ethyl acetate} =20:1).

The fluorescent probe for methanol detection of the present invention can be used for rapid detection of methanol samples.

The beneficial effects of the invention are as follows: the fluorescent probe for methanol detection of the invention can selectively identify methanol, and has the characteristics of strong anti-interference and high sensitivity. Due to its special response principle, this probe has no obvious response to other alcohol compounds and can be used for the determination of methanol in alcohol. The filter paper soaked with the probe solution can be used as a methanol test paper for qualitative detection of methanol-containing samples.

Description of drawings

Fig. 1 is the selective fluorescence response map of methanol fluorescent probe 1 prepared in Example 2 to common solvents.

Figure 2 shows the fluorescence spectrum and linear range (R ² = 0.9788) of methanol fluorescent probe 1 (20 μM) prepared in Example 2 to different concentrations of methanol/water solution (2-220 μM).

Detailed ways

Example 1 Synthesis of Rhodamine Compounds

罗丹明化合物

Rhodamine Compounds

Weigh 3-diethylaminophenol (1.65 g, 10.00 mmol) into a 100 mL flask, add 20 mL of toluene dried over molecular sieves, and slowly stir at 60 °C until completely dissolved. Phthalic anhydride (1.50 g, about 10.13 mmol) was added, and the mixture was refluxed for 12 h under N ₂ protection. Adjust to pH ≤ 4 with dilute hydrochloric acid solution (ω = 20%) to obtain a purple-red precipitate. The product was filtered with suction, washed with distilled water, and dried to obtain 0.70 g of crude 4-diethylaminoketo acid with a yield of 42.50%.

Weigh 1 g (3.2 mmol) of crude 4-diethylaminoketo acid into a 100 mL flask, add 10 mL of methanesulfonic acid, stir at 50 °C until fully dissolved, add 0.4 g (3.80 mmol) of m-aminophenol, It was heated to 120 °C under the protection of N ₂ for 10 h. After the reaction was completed, it was cooled to room temperature, adjusted to pH≤5 with 0.1 M Na ₂ CO ₃ solution, and then fully extracted with ethyl acetate (3×20 mL). The organic phases were combined, dried with MgSO ₄ , filtered with suction, and evaporated under reduced pressure. Solvent, silica gel column chromatography (V _{dichloromethane} : V _methanol = 1:1) to obtain 0.40 g of red solid, yield 40.35%. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.03 (d, J = 7.1 Hz, 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) ¹³ C NMR (101MHz, CDCL ₃ ) 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, 100.53, 97.15, 44.60, 12.44.

Example 2 Synthesis of Probe 1

Weigh 0.150 g (0.30 mmol) of the rhodamine compound prepared in Example 1 into a 50 ml flask, add 10 mL of a 2.5 mol/L dilute hydrochloric acid solution, and stir well at room temperature. The flask was placed in a solid ice-water mixture with sodium chloride and stirred for 5 min. Weigh 0.024g (0.35 mmol) of NaNO ₂ solid and slowly add it to the reaction solution. After fully stirring for 20 min, add 0.044 g (0.32 mmol) of p-nitroaniline to react for 20 min. Finally, adjust the pH to medium with 1 mol/L NaOH solution. sex. The reaction solution was fully extracted with ethyl acetate (3×30 mL), and the organic phases were combined, fully dried with anhydrous MgSO ₄ , evaporated under reduced pressure to remove the solvent, and chromatographed on silica gel (V _{petroleum ether} : V _{ethyl acetate} =20 : 1), and vacuum-dried to obtain probe 1 0.038g with a yield of 23.50%. ¹ HNMR (400 MHz, CDCl ₃ ) 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). ¹³ C NMR (101 MHz, CDCL ₃ ) Δ169.56,153.04, 152.54, 142.34, 134.93, 132.57, 129.70, 127.95, 125.57,124.06, 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 (ESId): anal ₃₀ ⁺ ] _H25N5O5 : _536.1928 ; found: _536.1907 .

Example 3 Synthesis of Probe 2

Probe 2 0.056g was synthesized according to the method of Example 2, and the yield was 43.33%. ¹ H NMR (400 MHz, CDCl ₃ ) δ8.05 (d, J = 7.4 Hz, 1H), 7.66 (dt, J = 23.3, 7.4 Hz, 2H), 7.45 (d, J = 8.6 Hz, 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). ¹³ C NMR (101 MHz, CDCL ₃ ) Δ 169.71, 153.11, 152.90, 152.73,149.65, 145.18, 144.83, 131.92, 129.56, 128.93, 127.24.96, 124.06, 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 ( ^ESId2 ): for C. ₃ H0 _{al ClN4O3} : _525.1687 ; found: 525.1692.

Example 4 Synthesis of Probe 3

According to the method of Example 2, 0.081 g of probe 3 was synthesized, and the yield was 54.26%. ¹ H NMR (400 MHz, CDCl ₃ ) δ8.06 (d, J = 7.6 Hz, 1H), 7.66 (dt, J = 22.5, 7.3 Hz, 2H), 7.52 (d, J = 8.2 Hz, 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). ¹³ C NMR (101 MHz, CDCL ₃ ) Δ 169.71, 153.09, 152.91, 152.72,149.68, 145.42, 145.07, 134.84, 132.55, 129.91, 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 ( _ESId ): [M+H ⁺ H an] _25BrN4O3 : _569.1128 ; found: _569.1187 .

Example 5 Synthesis of Probe 4

According to the method of Example 2, 0.070 g of probe 4 was synthesized, and the yield was 46.67%. ¹ H NMR (400 MHz, CDCl ₃ ) δ8.05 (d, J = 7.5 Hz, 1H), 7.66 (dt, J = 23.1, 7.4 Hz, 2H), 7.41 (d, J = 7.8 Hz, 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). ¹³ C NMR (101 MHz, CDCL ₃ ) Δ 169.70, 153.19, 152.94, 152.73,149.64, 145.30, 144.20, 136.60, 129.84, 129.26, 128.91,127.27, 124.9272 , ^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. _31H28N4O3 : _{505.2234 ;} found: _505.2217 .

Example 6 Probe Performance Test

The probe 1 prepared in Example 2 was selected as an example for performance testing.

1) Fluorescence spectra of probe 1 prepared in Example 2 in different solvents. A certain amount of probe 1 stock solution (1 mM) was added to each solvent to make the final concentration of probe 1 20 μM. The obtained sample was subjected to fluorescence spectrum measurement, and the results are shown in Fig. 1 (excitation light wavelength 480 nm). It can be seen from Figure 1 that probe 1 has a good fluorescence response to methanol, a weak fluorescence response to ethanol, and no fluorescence response to other common solvents.

2) Fluorescence responses of probe 1 to methanol aqueous solutions with different concentrations. A certain amount of probe 1 stock solution (1 mM) was added to aqueous solutions of different methanol concentrations (2-220 μM), respectively, so that the final concentration of probe 1 was 20 μM. The obtained sample was subjected to fluorescence spectrum measurement, and the results are shown in Fig. 2 (excitation light wavelength 480 nm). It can be seen from Figure 2 that with the increase of methanol concentration, the fluorescence intensity of the probe gradually increases. In the range of 2-220 μM, the fluorescence intensity has a linear relationship with the methanol concentration (R ² =0.9788), so the probe can quantitatively detect methanol.

3) Detection of methanol by probe 1 test paper. Immerse the filter paper strip in Probe 1 stock solution (1 mM) for 10 seconds, then allow the filter paper strip to dry naturally to obtain methanol test paper. The ethanol solution with a methanol content of 0.1% was added dropwise to the test paper for 1 min, and the test paper turned pink. There is no obvious phenomenon when pure ethanol is dropped on the test paper. The above experiments show that the test paper can easily detect the trace amount of methanol in ethanol.

Claims (6)

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:

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.

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.

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