🍎 Anisidine value of oils and fats — reaction with p-anisidine, spectrometry at 350 nm

Food chemistry PN-EN ISO 6885

Overview

The standard specifies a method of determining the anisidine value of animal and vegetable oils and fats, which is a measure of the amount of the aldehydes present (mainly α,β-unsaturated ones) — the secondary products of the oxidation of a fat. The anisidine value is one hundred times the increase of the absorbance, measured at 350 nm in a 10 mm cell, of the solution tested after the reaction with p-anisidine under the conditions of the standard; it is dimensionless and refers to 1 g of the sample in 100 ml of the mixture of the solvent and the reagent. Milk and milk products (and the fat from them) are excluded from the scope. The fourth edition ISO 6885:2016 replaces the edition of 2006.

Unlike the peroxide value, which covers the unstable primary products of oxidation, the anisidine value testifies to the content of the aldehydes and ketones arising from the decomposition of the hydroperoxides, and for that reason it is a good parameter for assessing the oxidative processes taking place in fats. In assessing the oxidation of an oil, the total oxidation value is helpful — the Totox index = 2·PV + AV (PV — the peroxide value in meq O2/kg, AV — the anisidine value). In a review of the oxidative stability of edible fats (Bromatologia i Chemia Toksykologiczna 2011) there is given among other things an increase of the anisidine value of an oil during frying from 1.2 to 59.8 and of the Totox index from 3.91 to 67.85.

The size of the test portion (usually 0.4–4.0 g) is chosen according to the quality of the sample and the characteristics of the spectrometer so that the absorbance of the solution after the reaction falls within the range 0.2–0.8; otherwise the determination is repeated with a different test portion. Samples with a moisture content above 0.10 % are dried with anhydrous sodium sulfate (1–2 g per 10 g of sample).

Method principle

A test portion of the fat is dissolved in isooctane in a 25 ml volumetric flask. From this solution 5 ml are taken into two test tubes: to one 1 ml of glacial acetic acid is added (the unreacted solution, absorbance A0), to the other 1 ml of the anisidine reagent (the reacted solution, A1); a third test tube with 5 ml of isooctane and 1 ml of the reagent constitutes the blank (A2). The test tubes are kept for 8 min in the dark at 23 ± 3 °C, within the next 2 min the contents are transferred into a dry cell and after a total reaction time of 10 ± 1 min the absorbance is measured at 350 nm against isooctane. The anisidine value: AV = 100·Q·V/m · [1.2·(A1 − A2 − A0)], where V = 25 ml, Q = 0.01 g/ml, m is the mass of the test portion (g), and 1.2 is the factor correcting for the dilution by 1 ml of the reagent. The result is given to one decimal place. Repeatability r = 0.034·AV + 0.31, reproducibility R = 0.19·AV + 1.41 in the range 0–100.

Applications

• Assessment of the secondary products of oxidation (aldehydes) in vegetable oils and animal fats
• Quality control of refined oils, frying fats and fish oils
• Study of the changes of fat during frying, heating and storage
• Calculation of the Totox index together with the peroxide value
• Assessment of the oxidative stability of fats in scientific studies

Key parameters

Standard

Standard number

PN-EN ISO 6885:2016-04

Title (PL)

Oleje i tłuszcze roślinne oraz zwierzęce — Oznaczanie liczby anizydynowej

Title (EN)

Animal and vegetable fats and oils — Determination of anisidine value

Step-by-step procedure

1. Preparation of the sample

   Prepare the sample according to ISO 661. If the moisture content exceeds 0.10 %, add anhydrous sodium sulfate (1–2 g per 10 g of the thoroughly mixed sample, a solid fat at a temperature not higher than 10 °C above its melting point), mix and filter, avoiding access of moisture.

2. The anisidine reagent

   On the day of use dissolve 0.125 g of p-anisidine in glacial acetic acid in a 50 ml flask and make up to the mark, protecting it from strong light. Check the absorbance against isooctane; discard the reagent where the difference exceeds 0.2. Destroy the remains on the same day.

3. Solution of the sample

   Weigh into a 25 ml flask an appropriate mass of the sample (usually 0.4–4.0 g) to the nearest 1 mg; warm solid samples beforehand to 10 °C above their melting point. Dissolve in 5–10 ml of isooctane and make up to the mark.

4. The unreacted solution (A0)

   With a pipette transfer 5 ml of the solution of the sample into a test tube, add 1 ml of glacial acetic acid, close it and shake; keep it for 8 min in the dark at 23 ± 3 °C, within 2 min transfer it into a clean dry cell.

   ⏱ Time: 10 ± 1 min

5. The reacted solution (A1)

   To 5 ml of the solution of the sample add with a pipette 1 ml of the anisidine reagent, shake, 8 min in the dark at 23 ± 3 °C, transfer into a cell; the measurement after a total reaction time of 10 ± 1 min from the addition of the reagent.

   ⏱ Time: 10 ± 1 min

6. The blank (A2)

   To 5 ml of isooctane add 1 ml of the anisidine reagent and proceed as in step 5.

7. Measurement

   Zero the spectrometer with isooctane at 350 nm and measure against isooctane the absorbances A1, A0 and A2. If A1 does not fall within the range 0.2–0.8, repeat the determination with a different test portion; if A2 exceeds 0.2, purify the p-anisidine and prepare a fresh reagent.

8. Calculation and test report

   AV = 100·Q·V/m · [1.2·(A1 − A2 − A0)] with V = 25 ml and Q = 0.01 g/ml; give it to one decimal place. Optionally Totox = 2·PV + AV. In the test report: the identification of the sample, the method of sampling, the reference to ISO 6885, the departures, the result.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• p-Anisidine is toxic — avoid contact with the skin, work in gloves and under a fume hood, prepare the minimum amount of reagent and destroy the remains on the same day
• Isooctane is flammable, glacial acetic acid corrosive — away from sources of ignition, protective goggles
• Pipetting only with a suction device

Frequently asked questions