The amount of test sample used depends on the presumed gossypol content of the sample. It is preferable to work with a small test sample and a relatively large aliquot part of the filtrate, so as to obtain sufficient gossypol for precise photometric measurement to be possible. For the determination of free gossypol in cottonseed, cottonseed meal and cottonseed cake, the test sample shall not exceed 1 g; for compound feed, it may be as much as 5 g. A 10 ml aliquot part of filtrate is suitable in most cases; it shall contain 50 to 100 μg of gossypol. For the determination of total gossypol, the test sample shall be between 0,5 and 5 g, that a 2 ml aliquot part of filtrate will contain 40 to 200 μg of gossypol.
The analysis shall be carried out at a room temperature of about 20 oC.
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.
Place a test sample containing 1 to 5 mg of gossypol in a 50 ml graduated flask and add 10 ml of solvent B (3.3). At the same time, prepare a blank test, placing 10 ml of solvent B (3.3) in another 50 ml graduated flask. Heat the two flasks for 30 minutes over a boiling water bath. Cool to room temperature and make the contents of each flask up to volume with the propan-2-ol-hexane mixture (3.1). Homogenise and leave to settle for 10 to 15 minutes, then filter and collect the filtrates in ground-necked flasks.
Pipette 2 ml of the sample filtrate into each of two 25 ml graduated flasks, and 2 ml of the blank test filtrate into each of two other 25 ml flasks. Make the contents of one flask from each series up to 25 ml with the propan-2-ol-hexane mixture (3.1). These solutions will be used as reference solutions.
Add 2 ml of aniline (3.4) to each of the other two flasks. Heat for 30 minutes over a boiling water bath to develop the colour. Cool to room temperature, make up to 25 ml with the propan-2-ol-hexane mixture (3.1), homogenise and leave to stand for one hour.
Determine the optical density as indicated in 5.2 for free gossypol. From this value calculate the total gossypol content as indicated in 6.