Marine lipids
Introduction
Centrifugal partition chromatography (CPC) is a liquid-liquid separation method relating to the partition of compounds between two immiscible phases of a solvent system, kept in contact in the chromatography column. One of these liquid phases, called the stationary phase, is
held in the instrument by centrifugal force. The other phase, called the
mobile phase, moves through the column. The separation of compounds
is performed according to the affmity of the molecule for one or the other
phase.
CPC is a powerful method which is emerging now in several areas,
including lipochemistry. For example, free fatty acids extracted from
the microalgae Skeletonema costatum have been separated (Bousquet et al.,
1994). This method was applied to the separation of ethyl esters of
EPA and DHA (Du et al., 1996). It was possible to obtain a mixture
EPA/DHA with the proportions of 56.4% and 39-3% using the system
hexane-dichloromethane-acetonitrile (5:1:4 v/v/v). Unfortunately, these
two esters were not resolved with this solvent system because they had
a low partition coefficient under these experimental conditions. Glycolipids, both acidic and neutral, were also submitted to CPC separation
(Otsuka et al., 1983) with the solvent mixture chloroform-benzenemethanol-water (50:25:65:30). This solvent system gave satisfactory
results for the separation of globoside 1 from human erythrocytes. The
separation of phospholipids was decribecl by Baudimant et al., 1996.
Materials and methods
Materials
Solvents of analytical grade were provided by C. Erba (Nanterre, France)
or Merck (Nogent-sur-Marne, France), silica gel, phospholipidic standards,
phospholipase A2 and specific revelators by Sigma (Saint-Quentin
Fallavier, France).
Fish oil was produced from fishing by-products in our laboratory.
Extraction of phospholipids
Total lipids were extracted from fish oil by the method of Bligh &
Dyer (1959). Fractionation of the various classes of lipids (neutral lipids,
glycolipids and phospholipids) was performed by adsorption chromatography on silica gel (silica gel 60, particle size 63-200 µm, 70-230pm
mesh, Merck, Nogent-sur-Marne, France), a method adapted from
Rouser et al. (1976). The phospholipids recovered with methanol
were evaporated under nitrogen and quantified to be expressed as
percentage of total lipids.
Centrifugal partition chromatography
Centrifugal partition chromatography (CPC) was performed on an
hydrodynamic apparatus, the Kromaton® III (SEAB, Villejuif, France).
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Introduction
Centrifugal partition chromatography (CPC) is a liquid-liquid separation method relating to the partition of compounds between two immiscible phases of a solvent system, kept in contact in the chromatography column. One of these liquid phases, called the stationary phase, is
held in the instrument by centrifugal force. The other phase, called the
mobile phase, moves through the column. The separation of compounds
is performed according to the affmity of the molecule for one or the other
phase.
CPC is a powerful method which is emerging now in several areas,
including lipochemistry. For example, free fatty acids extracted from
the microalgae Skeletonema costatum have been separated (Bousquet et al.,
1994). This method was applied to the separation of ethyl esters of
EPA and DHA (Du et al., 1996). It was possible to obtain a mixture
EPA/DHA with the proportions of 56.4% and 39-3% using the system
hexane-dichloromethane-acetonitrile (5:1:4 v/v/v). Unfortunately, these
two esters were not resolved with this solvent system because they had
a low partition coefficient under these experimental conditions. Glycolipids, both acidic and neutral, were also submitted to CPC separation
(Otsuka et al., 1983) with the solvent mixture chloroform-benzenemethanol-water (50:25:65:30). This solvent system gave satisfactory
results for the separation of globoside 1 from human erythrocytes. The
separation of phospholipids was decribecl by Baudimant et al., 1996.
Materials and methods
Materials
Solvents of analytical grade were provided by C. Erba (Nanterre, France)
or Merck (Nogent-sur-Marne, France), silica gel, phospholipidic standards,
phospholipase A2 and specific revelators by Sigma (Saint-Quentin
Fallavier, France).
Fish oil was produced from fishing by-products in our laboratory.
Extraction of phospholipids
Total lipids were extracted from fish oil by the method of Bligh &
Dyer (1959). Fractionation of the various classes of lipids (neutral lipids,
glycolipids and phospholipids) was performed by adsorption chromatography on silica gel (silica gel 60, particle size 63-200 µm, 70-230pm
mesh, Merck, Nogent-sur-Marne, France), a method adapted from
Rouser et al. (1976). The phospholipids recovered with methanol
were evaporated under nitrogen and quantified to be expressed as
percentage of total lipids.
Centrifugal partition chromatography
Centrifugal partition chromatography (CPC) was performed on an
hydrodynamic apparatus, the Kromaton® III (SEAB, Villejuif, France).
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