2.5.1.3. Carotenoids separation. About 20 ml distilled petroleum
ether was placed in a 250 ml separating funnel wrapped with black
cloth. The coloured acetone extract was carefully added to the petroleum
ether through a funnel, gently flowing down the wall of
separating funnel. The vacuum flask was rinsed with 1–2 ml acetone.
Then, 150 ml distilled water was added to the separating funnel
(flowing down the funnel wall), the mixture was left
undisturbed for 5–10 min to allow for separation into organic
and aqueous layers. The aqueous layer, containing acetone, was
discarded. This step was repeated for 3–4 times with 100 ml distilled
water added each time until the residual acetone was
removed. The petroleum ether extract in the separating funnel
was collected, through a funnel containing small amount of anhydrous
sodium sulfate on a filter paper, into a 25 ml volumetric flask
covered with aluminum foil. The separating funnel was rinsed with
about 2 ml petroleum ether using a pipette.
The eluent volume was made up to 25 ml with petroleum ether.
The flask was capped and
gently mixed. For determination of the carotenoids, absorbance of
the orange-coloured eluent was measured at 450 nm in a spectrophotometer
(UV-1601, UV-Visible, Shimadzu, Tokyo, Japan).
All the preparative and extraction procedures were performed in
dim light and/or excluded light as described.