3.2.6. Ethanol (de-emulsifying agent)
Ethanol was used to prevent emulsion formation between
hydrophobic and hydrophilic phase. The de-emulsifying agent
was necessary as milk has both hydrophobic as well as hydrophilic
phase and also emulsifying agents (phospholipids, amphoteric proteins
etc.). Ethanol prevented emulsion formation by which vitamin
D2 was effectively separated in hydrophobic phase and less
water came with extraction solvent, which made the following
evaporation step easier. 100, 150 and 200 ll ethanol were added
to extraction tubes. 200 ll ethanol gave clear separation between
the hydrophobic and non hydrophilic layers in comparison to
100 and 150 ll ethanol.