The most popular extraction procedure is that of Folch (Folch et al., J Biol Chem 1957, 226,
497):
Folch method
The tissue is homogenized with
chloroform/methanol (2/1) to a final volume 20 times the volume of the tissue sample (1 g
in 20 ml of solvent mixture). After dispersion, the whole mixture is agitated during
15-20 min in an orbital shaker at room temperature.
The homogenate is either filtrated (funnel with a folded filter paper) or centrifuged to
recover the liquid phase.
The solvent is washed with 0.2 volume (4 ml for 20 ml) of water
or better 0.9% NaCl solution. After vortexing some seconds, the mixture is centrifuged at
low speed (2000 rpm) to separate the two phases. Remove the upper phase by siphoning and
kept it to analyze gangliosides or small organic polar molecules. If necessary (need of
removing labelled molecules...), rinse the interface one or two times with methanol/water
(1/1) without mixing the whole preparation.
After centrifugation and siphoning of the upper phase, the lower chloroform phase containing lipids is evaporated under vacuum in a rotary evaporator or under a nitrogen stream if the volume is under 2-3 ml.
COMMENTS :
It must be emphasized that the method of tissue disruption affects the
concentrations of extracted lipids. Therefore, for poorly explored tissues, the
analyst must be careful in comparing various methods of tissue disruption.
As an example, it was demonstrated that better recoveries of liver lipids are
obtained extracting 150 mg of liver tissue with 30 second sonication (or a 2 min homogenization), followed by a
2h shaking in chloroform/methanol reagent and a 12h incubation in chloroform. A
drying time of 6h must be observed before weighing to get reliable lipid amounts
(Ametaj BN et al. J Agric Food Chem 2003, 51, 2105).
For the determination of total lipids, it must be pointed out that
precautions have to be taken against including in the determination any
non-lipid material that may be have become entrained. To avoid overestimating
with gravimetry, care has to be taken to wash the extract properly, to evaporate
all solvent from the extract, and to minimize any contact with oxygen.
Unsaturated lipids were shown to gain as much as 3.5% in mass due to adsorbtion
of oxygen.