Division 5.2 Determination of free gossypol

Place the test sample in a ground-necked 250 ml flask, the bottom of the flask having been covered with crushed glass. Using a pipette, add 50 ml of solvent A (3.2), stopper the flask and mix for one hour in the mixer. Filter through a dry filter and collect the filtrate in a small ground-necked flask. During filtration, cover the funnel with a watch glass.

Pipette identical aliquot parts of filtrate containing 50 to 100 μg of gossypol into each of two 25 ml graduated flasks (A and B). If necessary, make up the volume to 10 ml with solvent A (3.2). Then make the contents of flask (A) up to volume with the propan-2-ol-hexane mixture (3.1). This solution will be used as a reference solution against which to measure the sample solution.

Pipette 10 ml of solvent A (3.2) into each of two other 25 ml graduated flasks (C and D). Make the contents of flask (C) up to volume with the propan-2-ol-hexane mixture (3.1). This solution will be used as a reference solution against which to measure the blank test solution.

Add 2 ml of aniline (3.4) to each of flasks (D) and (B). Heat for 30 minutes over a boiling water bath to develop the colour. Cool to room temperature, make up to volume with the propan-2-ol-hexane mixture (3.1), homogenise and leave to stand for one hour.

Determine the optical density of the blank test solution (D) by comparison with the reference solution (C), and the optical density of the sample solution (B) by comparison with the reference solution (A), in the spectrophotometer at 440 nm using 1 cm glass cells.

Subtract the optical density of the blank test solution from that of the sample solution (= corrected optical density). From this value calculate the free gossypol content as indicated in 6.