Methyl 3-amino-4-methylthiophene-2-carboxylate determination of the content

Preparation of methyl 3-amino-4-methylthiophene-2-carboxylate

Take 8.7g (0.05 mol) 3-carbonyl-4-methyl-tetrahydrothiophene-2-carboxylic acid methyl ester, add a certain amount of acetonitrile, stir and heat to reflux, then add 3.5g (0.05 mol) hydroxylamine hydrochloride to react. The progress of the reaction was monitored by HPLC. After the reaction was completed, a purple solution was obtained, which was concentrated under reduced pressure to remove acetonitrile, 50 ml of ethyl acetate was added, and 10 ml of concentrated hydrochloric acid was added with stirring, resulting in a large amount of purple precipitate. Suction filtration, the filter cake was washed with a small amount of ethyl acetate. The resulting filter cake was dissolved in 50 ml of water, adjusted to pH 10.5 with ammonia, and then extracted with ethyl acetate (2×30 ml), the organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain a yellow solid. Dry under vacuum to obtain methyl 3-amino-4-methylthiophene-2-carboxylate, which is recrystallized from absolute ethanol.

How about the Determination of the content of methyl 3-amino-4-methylthiophene-2-carboxylate?

1. The preparation of the solution is accurately weighed

Methyl 3-amino-4-methylthiophene-2-carboxylate reference substance 10 mg, placed in 10 ml volume in the bottle, dissolve with 1 ml acetonitrile, and then dilute to the mark with a solution (A solution) containing 0.6 g phosphoric acid (84-85%) in 100 ml water to obtain a stock solution with a sample concentration of 1 g:L’. Accurately measure 0.5, 1, 2, 3, and 5ml of the stock solution, put them in a 10 ml volumetric flask, add solution A to the mark, shake well, and get the concentration respectively 50, 100, 200, 300, 500 mg: L” ‘Working solution.

2. Determination of UV absorption wavelength

Take a working solution with a concentration of 100 mg:L’, take solution A as a blank, and set it at 200~ scan it in the 400 nm wavelength range. As a result, methyl 3-amino-4-methylthiophene-2-carboxylate has a maximum absorption peak at 254 nm.

3. Preparation of HPLC standard curve

Chromatographic conditions: chromatographic column, Phenomenex Luna Ci8 column (4.6 mmx200 mm, 5 um); mobile phase, acetonitrile-water=30-70; detection wavelength, 254 nm; flow rate, 1.00 ml:min; column temperature: 25 °C.

Precisely measure each 10μl of the working solution and inject it into the HPLC, inject 3 times in parallel, and record the chromatogram. Take the concentration (X, mg:L) as the abscissa and the peak area (Y) as the ordinate, perform linear regression, and find the linear regression equation.

4. Determination of the content of methyl 3-amino-4-methylthiophene-2-carboxylate

Weigh a sample of methyl 3-amino-4-methylthiophene-2-carboxylate for approximately 10 mg, placed in a 10 ml volumetric flask, add 1 ml of acetonitrile to dissolve the sample, and then use A solution to make the volume. Accurately measure 2 ml of this solution and place it in a 10 ml volumetric flask, and use A solution to make the volume constant. Precisely measure 10 μl of the sample solution and inject it into the HPLC, inject 3 times in parallel, and record the chromatogram.

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