Food Lipids: Chemistry, Nutrition, and Biotechnology

a ternary system consisting of chloroform, methanol, and water in various proportions,
depending on the moisture content of the sample [9]. The method of Bligh
and Dyer [9] specifically recognizes the importance of water in the extraction of
lipids from most tissues and also plays an important role in purifying the resulting
lipid extract. A typical Folch procedure uses a solvent-to-sample ratio of 2:1 (v/w)
with a mixture of chloroform and methanol (2:1, v/v) in a two-step extraction. The
sample is homogenized with the solvent and the resultant mixture filtered to recover
the lipid mixture from the residue. Repeated extractions are usually carried out,
separated by washings with fresh solvent mixtures of a similar composition. It is
usually accepted that about 95% of tissue lipids are extracted during the first step.
In this method, if the initial sample contains a significant amount of water, it may
be necessary to perform a preliminary extraction with 1:2 (v/v) chloroform–methanol
in order to obtain a one-phase solution. This extract is then diluted with water or a
salt solution (0.08% KCl) until the phases separate and the lower phase containing
lipids is collected. Bligh and Dyer [9] uses 1:1 (v/v) chloroform–methanol for the
first step extraction and the ratio is adjusted to 2:1 (v/v) in the alternate step of
extraction and washing. The original procedure of Folch or of Bligh and Dyer uses
large amounts of sample (40–100 g) and solvents; therefore, the amounts may be
scaled down when a small amount of sample is present or for routine analysis in the
laboratory. Hence, Lee and coworkers [11] have described a method that uses the
same solvent combination, but in different proportions, based on the anticipated lipid
content of the sample. According to this method, chloroform–methanol ratios of
2:1 (v/v) for fatty tissues (>10% lipid) and 1:2 (v/v) for lean (