UV-1200 UV spectrophotometer for the determination of bacteria, thiophanate-methyl and carbendazim - Master's thesis - Dissertation

Determination of bacteria, thiophanate-methyl and carbendazim by ultraviolet spectrophotometer

Key words: thiophanate-methyl; carbendazim; bacteria; ultraviolet spectrophotometer; US instrumentation UV-1100; UV-1200 Measurement of thiophanate-methyl and carbendazim content for thiophanate-methyl thiophanate-methyl (also known as thiophanate-methyl) is a broad-spectrum systemic fungicide widely used for prevention and treatment. Ring disease and black rot of fruit and black mold and green mold during storage. However, due to the illegal operation of the manufacturer and the sales department, the content of thiophanate-methyl in the fruit exceeds the national standard (GB14870-94), and the maximum residue is not more than 0.5 mg per kilogram. In order to prevent the fruit and processed products of high-residue pesticides from being listed, the state requires that the content of thiophanate-methyl is determined by gas chromatography-mass spectrometry according to the national standard SN0162-92 before sale. However, the instrument is expensive and the operation technology is complicated. To this end, a simple, accurate, reliable, general laboratory testable UV spectrophotometer is introduced. Measurement of thiophanate-methyl and carbendazim content a. Preparation of standard stock solution and use solution Accurately weigh 50 mg of thiophanate-methyl WP, put it into a beaker, dissolve it with chloroform, and bring to volume 50ml. Accurately absorb the standard solution of 10·00 ml of thiophanate-methyl from the solution, transfer it to a 100 ml volumetric flask, and dilute to the mark with chloroform to prepare a standard solution of thiophanate-methyl. Also accurately weigh 50 mg of carbendazim into a 50 ml volumetric flask, dissolve it with 1 mol/L hydrochloric acid solution and dilute to volume. Then accurately absorb 10·00ml carbendazim standard stock solution, transfer it to a 100ml container bottle, and dilute to the mark with 1mol/L hydrochloric acid solution to prepare carbendazim standard use solution. b. Extraction and Separation of Samples to be Tested Randomly take fruit samples, wash off the sediment with water, dry or blot the water on the surface of the fruit with absorbent paper. The fruit is cut into two equal portions, and the pulp portion is taken and placed in a dry tissue masher or mortar to break and grind. Weigh 50g of puree sample, add 50ml of methanol, centrifuge with a high-speed centrifuge (6000 rpm) for 10 minutes, and transfer the supernatant to the beaker. The precipitate was stirred with 20 ml of methanol, and then added with water for 10 m, 1 for 10 minutes, and centrifuged again at high speed for 10 minutes. The supernatant was mixed into a beaker containing the filtrate, and a portion of the methanol was blown off with a hot air stream on a water bath at 80 ° C, poured into a separatory funnel, and 10 ml of a sodium chloride solution (30 ml) and 25 ml of petroleum ether were added, and the mixture was shaken for 2 minutes. After that, shake with 25 ml of petroleum ether for 2 minutes to fully extract the drug solution to be tested. The petroleum ether was removed, hydrochloric acid was added to increase the acidity to pH 1-2, and extracted twice with dichloromethane (25 m each time, 1 for 2 minutes). The extracts were combined twice, washed once with 25 ml of distilled water, and the methylene chloride layer was aspirated with a pipette tip and transferred to a dry separatory funnel for determination of thiophanate-methyl content. The washing liquid was combined into the water layer and used for the determination of carbendazim. c. Determination of residual toxicity 1 Draw a standard curve to accurately absorb 0·0, 0·1, 0·3, 0·5ml thiophanate-methyl standard use solution (equivalent to 0, 10, 30, 50μg thiophanate) Place them in a 30ml round bottom centrifuge, centrifuge to dry the solvent, add 10ml acetic acid-copper acetate solution and 2~5 glass beads, connect to the air condenser, and slowly heat the boiling on the alcohol lamp or micro electric furnace. After taking it off for a minute, the condensation tube and the round bottom centrifuge tube were washed with 20 ml of 1 mol/L hydrochloric acid for 2 minutes. The liquid was transferred to a 125 ml separatory funnel. Extract twice with dichloromethane, 10 ml each time. The methylene chloride layer was aspirated with a pipette tip and transferred to a spare dry separatory funnel. The above acid solution was neutralized with 2 mol/L sodium hydroxide, and the pH was adjusted to 6·0 to 6·5 (the amount of the lye was 25 ml), and the neutralized solution was extracted twice with dichloromethane for 20 m each time. The two extracts were combined, and the separatory funnel was washed with 10 ml of distilled water. After standing to separate the layers, the dichloromethane layer was combined into another separatory funnel containing a dichloromethane layer. Accurately add 10 ml of 1 mol/L hydrochloric acid, extract again for 5 minutes, and let stand for about 10 minutes. After layering, the acid extract was poured into a 1 cm quartz two-color cup, the zero point of the spectrophotometer was adjusted with 1 mol/L hydrochloric acid, and the absorbance of 0-50 μg of thiophanate-methyl standard solution was measured at a wavelength of 282 nm (A282). ). The standard curve of thiophanate-methyl was prepared by taking the A282 value as the ordinate and the content of thiophanate-methyl as the abscissa. Accurately absorb 0. 0, 0·1, 0·3, 0·5ml carbendazim standard use solution (equivalent to 0, 10, 30, 50μg carbendazim), put into the separatory funnel and add 20ml concentration to 1mol/ L hydrochloric acid was extracted twice with dichloromethane, 10 ml each time. The methylene chloride layer was aspirated with a pipette and transferred to a dry separatory funnel. The acid solution was neutralized to a pH of 6.00 to 6.5 with a 2 mol/L sodium hydroxide solution, and extracted twice with dichloromethane for 20 m each time, and the extract was washed once with 10 ml of distilled water. After standing to separate the layers, the two dichloromethane layers were combined, and 10 ml of hydrochloric acid having a concentration of 1 mol/L was accurately added, and acid extraction was further carried out for 5 minutes. Let stand for 10 minutes. After stratification, the acid extract was poured into a 1 cm quartz two-color cup, and the zero point of the spectrophotometer was adjusted with 1 mol/L hydrochloric acid. The absorbance value (A282) of 0 to 50 μg of carbendazim standard solution was measured at a wavelength of 282 nm. The standard curve of carbendazim was plotted with the A282 value as the ordinate and the carbendazim content as the abscissa. 2 Determine the content of thiophanate-methyl and carbendazim in the fruit. Take the dichloromethane extract of the pulp, and then dry it or heat it to dry. Dissolve the residue in 10 ml of acetic acid-copper acetate solution and transfer to a 30 ml round bottom. In the centrifuge, add 2~5 glass beads, connect the air condenser tube, slowly boil for 30 minutes with alcohol lamp or micro electric furnace, and wash the condensation tube and circle from the top of the condenser tube with 20ml hydrochloric acid with a concentration of 1mol/L hydrochloric acid. The bottom centrifuge tube was applied for 2 minutes. The liquid was transferred to a 125 ml separatory funnel and extracted twice with dichloromethane for 10 ml each time. The methylene chloride layer was aspirated with a pipette tip and transferred to a spare dry separatory funnel. The above acid solution was neutralized with 2 mol/L sodium hydroxide, and the pH was adjusted to 6·0 to 6·5 (the amount of the alkali solution was 25 ml). The neutralized solution was extracted twice with dichloromethane for 20 m each time. The two extracts were combined together, and the separatory funnel was washed with 10 ml of distilled water. After standing to separate the layers, the dichloromethane layer was combined into another. In a separatory funnel containing a dichloromethane layer, 10 ml of hydrochloric acid having a concentration of 1 mol/L was accurately added, and extracted again for 5 minutes, and allowed to stand for about 10 minutes. After stratification, the acid extract was poured into a 1 cm quartz two-color cup, and the spectrophotometer zero was adjusted with 1 mol/L hydrochloric acid. The absorbance of the sample was measured at a wavelength of 282 nm. The content of thiophanate-methyl in the sample was calculated in comparison with the standard curve of thiophanate-methyl. Take the carbendazim determination solution in the “Extraction and Separation of Samples to be Tested”, neutralize it to pH value of 6.00~6·5 with 2mol/L ammonium hydroxide solution, and extract twice with dichloromethane for 20m each time. The extract was washed once with 10 ml of distilled water, and after standing to separate the layers, the two dichloromethane layers were combined, and 10 ml of a hydrochloric acid having a concentration of 1 mol/L was accurately added, and acid extraction was further carried out for 5 minutes. After standing for 10 minutes, after layering, the acid extract was poured into a 1 cm quartz two-color cup, and the zero point of the spectrophotometer was adjusted with 1 mol/L hydrochloric acid. The absorbance of the sample was measured at a wavelength of 282 nm. The content of carbendazim in the sample was calculated in comparison with the standard curve of carbendazim. The measurement result is calculated by the following formula: X = V1 × Cm × V2 × 100 where V1 is the total number of milliliters of the extraction solvent; m is the sample weight (g); V2 is the number of milliliters of the extraction solvent used in the measurement; C is equivalent At the standard concentration (mg); X is the content of thiophanate-methyl (mg/kg) in the sample. (Note: The whole process of extraction with petroleum ether and dichloromethane, whether added or removed, must be accurate and fast. When the residual amount of thiophanate-methyl is too high, the amount of sample can be reduced or the dilution factor can be increased.) Key words: thiophanate-methyl; carbendazim; bacteria; ultraviolet spectrophotometer; US instrumentation UV-1100; UV-1200