[0037] Combine below figure 1 with 2 As shown, the synthesis process of the reagent for detecting hair water and soy sauce in this embodiment is introduced in detail.
[0038] In this example, by first synthesizing 1,3,5,7-tetramethyl-8-phenyl-(2-nitro)-boron difluoride dipyrromethene (TMPB-oN), then synthesizing 1, 3,5,7-tetramethyl-8-phenyl-(2-amino)-boron difluoride dipyrromethene (TMPB-oA), and finally obtained the test reagent of the present invention.
[0039] Specifically, first, accurately weigh 1.06 g (7.0 mmol) of o-nitrobenzaldehyde and dissolve it in 500 mL of dry CH 2 Cl 2.
[0040] Next, under the protection of nitrogen gas, 1.44 mL (14.0 mmol) of 2,4-dimethylpyrrole was slowly added dropwise to the obtained solution while stirring. After that, 1 drop of trifluoroacetic acid was dropped into the solution, and the resulting solution was sealed and kept at 25° C. and stirred overnight until it was detected by thin-layer chromatography that the o-nitrobenzaldehyde spots had disappeared.
[0041] Next, 4.0 mL of triethylamine was added, and the resulting solution was stirred at 25° C. for 10 min, and then 4.8 mL of boron trifluoride ether was added and stirred at room temperature overnight. Subsequently, the resulting mixture was concentrated to about 150mL, using saturated NaHCO 3 The solution washes the concentrate (40 mL×3). The organic phase was dried with anhydrous sodium sulfate, the solvent was removed, and purified by silica gel column chromatography (eluent: cyclohexane: CH 2 Cl 2 =30:70) to obtain 0.91 g of orange-red solid (yield: 35.3%). 1 H NMR(CDCl 3 , 300MHz): 1.36 (s, 6H), 2.56 (s, 6H), 5.99 (s, 2H), 7.47 (d, 1H), 7.69 (t, 1H), 7.78 (t, 1H), 8.19 (d, 1H).
[0042] The orange solid obtained is 1,3,5,7-tetramethyl-8-phenyl-(2-nitro)-boron difluoride dipyrromethene (TMPB-oN), which is then synthesized using TMPB-oN 1, 3,5,7-Tetramethyl-8-phenyl-(2-amino)-boron difluoride dipyrromethene (TMPB-oA).
[0043] Specifically, the obtained 0.91g (2.46mmol) TMPB-o-N was dissolved in 20mL CH 2 Cl 2 And 20 mL of absolute ethanol, 0.1 g of 10% Pd/C was added, hydrogen was introduced under normal pressure under stirring at room temperature for catalytic reduction, and the progress of the reaction was monitored by thin layer chromatography. After about 8h, the reaction was completed, the Pd/C was quickly removed by suction filtration, and the filtrate was rotary evaporated to remove the solvent and drained to obtain 0.79 g (yield: 94.5%) of crude TMPB-o-A, which was used directly without further purification and characterization.
[0044] Next, dissolve the obtained 0.79g TMPB-o-A in 100mL glacial acetic acid, add 0.27g (2.76mmol) maleic anhydride under stirring, stir for 2h, remove the solvent under reduced pressure, and purify by silica gel column chromatography (eluent: CH 2 Cl 2 :MeOH=19:1), 0.93g of pure orange product (yield: 91.3%) was obtained. 1 H NMR(CDCl 3 , 300MHz): 1.42 (s, 6H), 2.49 (s, 6H), 6.00 (s, 2H), 6.27 (d, 1H), 6.42 (d, 1H), 7.27 (m, 1H), 7.40 (m, 1H), 7.56 (m, 1H), 8.30 (m, 1H), 8.53 (br, 1H).
[0045] The obtained product was dissolved in 150 mL of acetic anhydride, 0.2 g of sodium acetate was added, and the temperature was kept at 80° C. overnight. TLC monitored the reaction to be complete. Most of the solvent was removed from the mixed solution under reduced pressure, and the rest was poured into water and stirred until a solid precipitated out. Filtration, and the obtained crude solid product is purified by silica gel column chromatography (eluent: CH 2 Cl 2 ), the pure product TMPAB-o-M 0.52g (yield: 58.3%), its molecular structure is as image 3 Shown. 1 H NMR(CDCl 3 , 300MHz): 1.57 (s, 6H), 2.51 (s, 6H), 5.96 (s, 2H), 6.65 (s, 2H), 7.35-7.38 (m, 1H), 7.47-7.50 (m, 1H), 7.60-7.63 (m, 2H).
[0046] Through the above method, a test reagent for fluorescent reaction with cysteine in hair water soy sauce is obtained. Although cystine and cysteine usually coexist, in order to improve the inspection efficiency of the test reagent of the present invention, the test reagent of the present invention may also include a reducing agent. The reducing agent can be added to the soy sauce to be tested in advance.
[0047] The following introduces how to use the detection reagent prepared by the present invention to detect soy sauce samples.
[0048] Under normal circumstances, the TMPAB-o-M solid obtained above can be configured into a methanol or dimethyl sulfoxide solution with a predetermined concentration, such as 1.0×10 -4 M and 5.0×10 -4 M. Then take a certain amount of soy sauce samples to be tested. In order to enhance the detection effect, the soy sauce samples can be pretreated. The pretreatment may include using activated carbon or other adsorbent materials to adsorb macromolecules or other impurities in the soy sauce, and may also include diluting the soy sauce sample to a predetermined concentration. Then, an excessive amount of reducing agent was added to the soy sauce sample, so that the cystine in the soy sauce sample was converted to cysteine. Then, drop the prepared test reagent into the soy sauce sample to observe or measure the fluorescence reaction.
[0049] In this example, the Zhenji brand was purchased from the supermarket: Soy Sauce Premium Soy Sauce, Premium Soy Sauce Soy Sauce, Grade 1 Soy Sauce, Light Grade 2 Soy Sauce; Jiajia Brand Soy Sauce: Premium First Grade Fresh, Gold Label Grade 1 Light Soy Sauce, Haitian A variety of soy sauces such as the brand Weijixian premium soy sauce and Lee Kum Kee premium soy sauce are used as samples. All kinds of soy sauces are purchased from Zhongbai Supermarket. In addition, the inventor weighed 0.0011g of TMPAB-o-M solid and dissolved it in DMSO to make 2.0×10 -3 M stock solution for subsequent testing.
[0050] In addition, in order to adjust the pH value, a PBS buffer solution is also prepared in this embodiment. The preparation process is as follows: Weigh 4.00g NaCl, 0.10g KCl, 0.10g KH 2 PO 4 , 1.39g Na 2 HPO 4 ·12H 2 O, dissolved in 500mL secondary water, adjusted to the desired pH value with 1M HCl or NaOH on the pH meter. H 3 Cit-NaOH buffer solution consists of 0.10M H 3 Cit solution and 0.10M NaOH solution are mixed and adjusted to the desired pH value. This buffer is used to dilute reagents or samples.
[0051] When testing, add 10μL 2.00×10 to 1.5mL plastic EP tube in turn -3 M TMPAB-o-M stock solution and an appropriate amount of soy sauce sample to be tested were diluted to 300 μL with a pH=7.40 PBS buffer solution. The mixture was reacted at 25°C for 8 minutes, diluted with liquid chromatography mobile phase and then directly injected for analysis. Directly observe the fluorescence response of the sample and perform a quantitative test on the fluorescence response.
[0052] In this example, a variety of soy sauce samples were tested. Specifically, the present invention takes the above soy sauce samples separately, dilutes them to a certain concentration, and then divides them into two parts, one part is mixed with hair lotion, and the other part is not mixed with hair lotion. Then, a comparative test was carried out on the soy sauce mixed with hair water and the soy sauce without hair water. The applicant found that the soy sauce without hair water basically has no fluorescence, while the soy sauce mixed with hair water has strong fluorescence.
[0053] Therefore, based on the above experiments, it can be proved that the reagent prepared by the method of the present invention can effectively detect hair water and soy sauce. Because the detection reagent of the present invention is simple and convenient to use, soy sauce can be sampled and inspected anytime and anywhere in the market, so as to effectively supervise the hair water soy sauce and prevent it from entering the market.
[0054] Although the principle of the present invention has been described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above-mentioned embodiments are merely explanations of exemplary implementations of the present invention and do not limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Without departing from the spirit and scope of the present invention, any obvious changes such as equivalent changes or simple replacements based on the technical solutions of the present invention shall fall into the present invention. Within the scope of protection.
