Food Lipids: Chemistry, Nutrition, and Biotechnology

E. Removal of Nonlipid Contaminants from Lipid Extracts and
Other Practical Considerations
Removal of nonlipid contaminants from the lipid extract is necessary since most of
the solvents employed also dissolve significant amounts of oil-soluble flavors, pigments,
sugars, amino acids, short chain peptides, inorganic salts, and urea. The nonlipid
matter must be removed prior to gravimetric determination of total lipids in
order to prevent contamination during subsequent fractionation of the total extract.
In chloroform–methanol extract the commonly used method for removing nonlipid
contaminants includes washing with water or a diluted KCl solution (0.88%, w/v).
Use of salt solution has the advantage of preventing or minimizing the formation of
an intermediate phase. When chloroform–methanol (2:1, v/v) is used for extraction
of the sample, addition of water or diluted salt solution results in the formation of
a two-phase system, i.e., a lower phase consisting of chloroform–methanol–water
(86:14:1, v/v/v) and an upper phase consisting of the same, but in the ratio of 3:48:
47 (v/v/v). The lower phase composes about two-thirds of the total volume and
contains the lipid components, but the upper phase retains the nonlipid contaminants.
However, more polar lipids, such as some phospholipids and glycolipids and all
gangliosides, may remain in the upper phase [4,47]. Nonlipid contaminants may also
be removed partly or completely by evaporation of the lipid extract to dryness in
vacuo or under nitrogen and then reextracted with a nonpolar solvent, such as hexane.
In the Bligh and Dyer [9] method the sample is homogenized with chloroform
and methanol in such proportions that a miscible system is formed with water in the
sample. Dilution of the homogenate with chloroform and water separates it into two
layers; the chloroform layer contains all the lipids and the methanol–water layer
contains all the nonlipid matter. A purified lipid extract could be obtained by isolating
the chloroform layer. Traces of moisture can be removed by passing the chloroform
extract through a bed of anhydrous sodium sulfate.
Removal of nonlipid contaminants by liquid–liquid partition chromatography
on a dextran gel was introduced by Wells and Dittmer [48]. This is done by passing
the crude lipid extract through a Sephadex G-25 column (packed in the upper phase
of chloroform–methanol–water, 8:4:3, v/v/v or ‘‘Folch wash’’). Lipids free of contaminants
would be eluted rapidly from the column by employing the lower phase
of the Folch wash. Gangliosides and non-lipids are retained in the column and can
be recovered by washing with the upper phase of Folch wash and at the same time
regenerating the column [49].
Use of predistilled solvents for lipid extraction is advisable since all solvents
contain small amounts of lipid contaminants. Use of plastic containers and non-
Teflon apparatus should also be avoided as plasticizers may leach out to the lipid
extract. To prevent autoxidation of unsaturated lipids it is advisable to add an antioxidant
(e.g., BHT) to the solvent (at a level of 50–100 mg/L). Furthermore, extraction
should be performed under an inert nitrogen atmosphere, and both tissue
and tissue extracts should be stored at �20�C under nitrogen, if possible. Most of
the methods described above are suitable for quantifying total lipid content of the
sample of interest. When high temperatures are involved in extraction the resulting
lipid extract is not suitable for further composition analysis. Folch method–based
extraction is usually the preferred procedure to obtain total lipids for further analysis.
Lipids are recovered from the chloroform layer by removal of solvent at low tem-
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