1. Lipid Determination in Aquatic Samples
13
light-scattering detector and its application to complex lipid samples (Christie,
1996; Heinz, 1996), this may soon be no longer true.
1.2.5. Determination of Fatty Acids and Carbon
Number Profiles
There are about 15-20 major fatty acids that can be readily recognized in most
samples. They differ in chain length and degree of unsaturation; certain ones
among them can be used as biomarkers of microorganisms in ecological studies
(Ahlgren et aI., 1992; Sargent et aI., 1987), and others are studied for the essential
nutritional role they play (Henderson, 1996).
Fatty acids are esterified in most biogenic lipid classes. They are present in
those that are termed acyl lipids but not in others such as hydrocarbons, ketones,
alcohols, and sterols. The fatty acids are released from the acyl lipid classes and
are re-esterified to methyl esters to make them amenable to gas chromatographic
(GC) analysis (Fig. 1.6). Boron trifluoride has been recommended as a catalyst for
the formation of fatty acid methyl esters by the American Oil Chemists' Society
(AOCS, 1989) in its official method, but other derivatization procedures are
available (Liu, 1994; Christie, 1989). Problems can occur with BF3 if abnormally
high concentrations are used (Morrison and Smith, 1964).
To perform fatty acid analyses, a variety of GCs are available; however, we
have generally used a Varian 3400 GC equipped with an autoinjector. Likewise, a
variety of capillary columns is available. The AOCS official method for marine
oils states that the column should be at least 25 m long, with a 0.20-0.35-mm
internal diameter, and it should be made of flexible fused silica. The method
recommends Carbo wax -20M or an equivalent such as SUPELCOWAX -10 for the
liquid phase coating the inner wall of the column; however, in my laboratory an
Omegawax 320 column (30 m, 0.32 mm i.d., 0.25-f.Lm film thickness; Supelco,
Inc.) has generally been used. This column was introduced in 1990 specifically for
use with official methods for polyunsaturated fatty acids. Recently, Supelco has
introduced a slightly lower polarity column, SPB-PUFA, which may be useful for
specific analyses or as a confirmational tool for analyses performed on other
columns. Similarly, Hewlett-Packard has introduced a midpolarity-phase column
(HP-225) that may be especially useful for rapid separation of hydroxy acids.
By using an Omegawax 320 column in our Varian GC, good analyses have been
obtained (Fig. 1.7), provided special attention is paid to solvent plug size in the
autoinjector and to oxygen in the carrier gas. For fatty acid analyses, the optimum
plug size was found to be 0.8 f.Ll, and the best carrier gas was hydrogen that had
been passed through an oxygen trap (S. Budge, Memorial University of Newfoundland, personal communication). With the use of hydrogen as a carrier gas,
extra attention has to be paid to leaks and to laboratory ventilation.
Standards are available from several sources for peak identification and quantitation. Polyunsaturated fatty acid mixtures from Supelco have been used for
peak identification in my laboratory. A very useful reference to aid in this process
is that of Ackman (1986). The areas under the peaks in the chromatograms should
13
light-scattering detector and its application to complex lipid samples (Christie,
1996; Heinz, 1996), this may soon be no longer true.
1.2.5. Determination of Fatty Acids and Carbon
Number Profiles
There are about 15-20 major fatty acids that can be readily recognized in most
samples. They differ in chain length and degree of unsaturation; certain ones
among them can be used as biomarkers of microorganisms in ecological studies
(Ahlgren et aI., 1992; Sargent et aI., 1987), and others are studied for the essential
nutritional role they play (Henderson, 1996).
Fatty acids are esterified in most biogenic lipid classes. They are present in
those that are termed acyl lipids but not in others such as hydrocarbons, ketones,
alcohols, and sterols. The fatty acids are released from the acyl lipid classes and
are re-esterified to methyl esters to make them amenable to gas chromatographic
(GC) analysis (Fig. 1.6). Boron trifluoride has been recommended as a catalyst for
the formation of fatty acid methyl esters by the American Oil Chemists' Society
(AOCS, 1989) in its official method, but other derivatization procedures are
available (Liu, 1994; Christie, 1989). Problems can occur with BF3 if abnormally
high concentrations are used (Morrison and Smith, 1964).
To perform fatty acid analyses, a variety of GCs are available; however, we
have generally used a Varian 3400 GC equipped with an autoinjector. Likewise, a
variety of capillary columns is available. The AOCS official method for marine
oils states that the column should be at least 25 m long, with a 0.20-0.35-mm
internal diameter, and it should be made of flexible fused silica. The method
recommends Carbo wax -20M or an equivalent such as SUPELCOWAX -10 for the
liquid phase coating the inner wall of the column; however, in my laboratory an
Omegawax 320 column (30 m, 0.32 mm i.d., 0.25-f.Lm film thickness; Supelco,
Inc.) has generally been used. This column was introduced in 1990 specifically for
use with official methods for polyunsaturated fatty acids. Recently, Supelco has
introduced a slightly lower polarity column, SPB-PUFA, which may be useful for
specific analyses or as a confirmational tool for analyses performed on other
columns. Similarly, Hewlett-Packard has introduced a midpolarity-phase column
(HP-225) that may be especially useful for rapid separation of hydroxy acids.
By using an Omegawax 320 column in our Varian GC, good analyses have been
obtained (Fig. 1.7), provided special attention is paid to solvent plug size in the
autoinjector and to oxygen in the carrier gas. For fatty acid analyses, the optimum
plug size was found to be 0.8 f.Ll, and the best carrier gas was hydrogen that had
been passed through an oxygen trap (S. Budge, Memorial University of Newfoundland, personal communication). With the use of hydrogen as a carrier gas,
extra attention has to be paid to leaks and to laboratory ventilation.
Standards are available from several sources for peak identification and quantitation. Polyunsaturated fatty acid mixtures from Supelco have been used for
peak identification in my laboratory. A very useful reference to aid in this process
is that of Ackman (1986). The areas under the peaks in the chromatograms should