The prepared crude extract was divided into two portions; one
portion (10 g) was poured into the glass vials to be tested as crude
methanol extract for antimicrobial activity, while the second portion
(100 g) was taken in a glass beaker for fractionation with different
solvents. The second portion was dissolved in water, poured into a
separatory funnel and then distilled petroleum ether was added into
it. The separatory funnel was shaken to separate the two phases as
petroleum ether being immiscible with water. Compounds soluble in
the upper petroleum ether phase (petroleum ether being lighter
than water) were collected and the lower aqueous phase was
extracted thrice with petroleum ether. All fractions of petroleum
ether were combined and poured into round bottom flask of rotary
evaporator and petroleum ether was isolated from the fraction
leaving behind semisolid petroleum ether fraction. The semisolid
petroleum ether fraction was dried in a China dish via water bath at
about 45°C and was stored in the glass vials until used. The same
process of fractionation was carried out for chloroform (heavier than
water), ethyl acetate (lighter than water) and butanol (lighter than
water), respectively resulting in chloroform, ethyl acetate and
butanol fractions. The lower aqueous phase at the end of the
process was taken and dried via rotary evaporator and water bath.