GB/T 24304-2024Animal and vegetable fats and oils - Determination of anisidine value (English PDF)

5.2 Isooctane (2,2,4-trimethylpentane). absorbance not exceeding 0.01 with water as a

reference in the wavelength range of 300 nm~380 nm.

5.3 p-Anethole (4-anisole). anhydrous, milky white crystals.

WARNING. p-Anisidine is toxic and shall be avoided in contact with skin.

Store p-anisidine in a brown bottle. Store in the dark at 0°C~4°C. p-Anisidine shall be milky white. If it turns gray or pink, purify it as follows.

Dissolve 4 g of p-anisidine in 100 mL of distilled water at 75°C. Add 0.5 g of sodium sulfite and 2 g of activated carbon. Stir for 5 min. Filter with medium-speed filter paper to obtain a clear filtrate. Cool the filtrate to 0°C. Maintain this temperature for at least

4 h to obtain p-anisidine crystals. Filter or vacuum filter the crystals. Wash with a small amount of distilled water at 0°C. Dry the purified p-anisidine crystals in a vacuum desiccator.

5.4 Glacial acetic acid. the water content by mass fraction is not greater than 0.1%.

5.5 Anisidine reagent. anisidine is toxic and unstable. Prepare only the minimum amount required for the analysis on the same day. For example, a 50 mL reagent can be prepared as follows. accurately weigh 0.125 g of p-anisidine, dissolve in glacial acetic acid, and dilute to volume in a 50 mL volumetric flask, avoiding direct sunlight.

Before use, measure the absorbance using isooctane as a blank control. If the absorbance is above 0.2, the reagent shall not be used. Under no circumstances shall the prepared reagent be used on the same day.

9.1 Preparation of test solution

Weigh an appropriate amount of the specimen (Chapter 8), accurate to 1 mg. Dissolve in 5 mL~10 mL of isooctane (5.2). Dilute to volume in a 25 mL volumetric flask. The solid specimen shall be preheated to above its melting point, but not exceeding 10°C below the melting point.

The sample mass is determined by the properties of the specimen and the spectrophotometer used, and ranges from 0.4 g~4.0 g. The absorbance of the specimen solution must not exceed the upper and lower limits of the spectrophotometer reading.

9.2 Determination of unreacted solution

Using a pipette (6.4), transfer 5 mL of the test solution (9.1) into a stoppered test tube (6.3). Using a pipette (6.4), add 1 mL of glacial acetic acid (5.4) solution. Stopper the tube and shake well. Incubate in the dark at 23°C ± 3°C for 8 min.

Transfer the solution to a clean, dry cuvette in a spectrophotometer within 2 min. The total reaction time, starting from the addition of the glacial acetic acid solution, is 10 min ± 1 min. Measure the absorbance according to 9.5.

9.3 Determination of the reaction solution

Using a pipette (6.4), transfer 5 mL of the test solution (9.1) into a stoppered test tube (6.3). Using a pipette (6.4), add 1 mL of anisidine reagent (5.5). Stopper the tube and shake well. Incubate in the dark at 23°C ± 3°C for 8 min.

Transfer the solution to a clean, dry cuvette in a spectrophotometer within 2 min. The total reaction time, starting from the addition of the anisidine reagent, is 10 min ± 1 min.

Measure the absorbance according to 9.5.

9.4 Preparation of blank test solution

Using a pipette (6.4), transfer 5 mL of isooctane (5.2) into a stoppered test tube (6.3).

Using a pipette (6.4), add 1 mL of anisidine reagent (5.5). Stopper the tube and shake well. Incubate in the dark at 23°C ± 3°C for 8 min.

Transfer the solution to a clean, dry spectrophotometer cuvette within 2 min. The total reaction time, starting from the addition of the anisidine reagent, is 10 min ± 1 min.

Measure the absorbance according to 9.5.

9.5 Determination of absorbance using a spectrophotometer

The spectrophotometer zero point was calibrated at 350 nm using isooctane (5.2). Using isooctane as a control, the absorbance of the following solutions is measured.

- A1.reaction solution (9.3);

- A0.unreacted solution (9.2);

- A2.blank test solution (9.4).

9.6 Absorbance range

If the absorbance of the reaction solution (9.3) A1 is not between 0.2~0.8, the sample weight needs to be adjusted and the determination repeated (9.2 to 9.4).

If the absorbance of blank solution A2 exceeds 0.2, p-anisidine needs to be purified, a freshly prepared anisidine reagent needs to be made, and the result shall be re-measured using the freshly prepared anisidine reagent.

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