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High-ResolutionAnalysesof FattyAcidMethyl EstersbyGasChromatography
Fatty acidmethyl esters (FAMEs) analysis is an important tool both in
characterizing fats andoils and indetermining the total fat content in
foods. Fats canbe extracted from amatrix, using a non-polar solvent,
and saponified toproduce salts of the free fatty acids.After derivatiz-
ing the free acids to form themethyl esters, themixture readily canbe
analyzedbygas chromatography (GC), due to the volatility and ther-
mal stabilityof theFAMEs. Gas chromatographyhas become an
important technique in fats andoils analysis because accurate results
canbe obtained for complex, aswell as simple, samplematrices.
FAMEs analyseswere among the first applications for gas chro-
matography, somany of theGCmethods originallywritten for
analysis of fats and oils described packed column technology.
Capillary columns offer significant advantages, however, including
more efficient separations.When analyzing fats and oilswith com-
plex fatty acid profiles, such as the
cis
and
trans
forms of polyun-
saturated fatty acids, higher efficiencies are needed to resolve the
individual components. Capillary columnswithCarbowax
®
-type
(polyethylene glycol) stationary phases typically are used for anal-
yses of saturated and unsaturated fatty acidmethyl esters, and bis-
cyanopropyl phases are used to resolve
cis
and
trans
isomers of
polyunsaturated components.
CreatingFAMEs
Lipids are normally extracted frommatrices using a non-polar sol-
vent, such as ether, and saponified to produce the free fatty acid
salts. The fatty acid salts then are derivatized to form the fatty acid
methyl esters, to increase volatility, improve peak symmetry,
decrease sample activity, and thus providemore accurate analytical
data. The official methods ofAOAC International
1
and theAmerican
Oil Chemists Society (AOCS)
2
both contain procedures for the
derivatization reaction, as does the European Pharmacopoeia.
3
In
general, the glycerides are saponified by refluxingwithmethanolic
sodium hydroxide. The esterification is effectedwith a reagent such
as boron trifluoride inmethanol and the FAMEs are extractedwith a
non-polar solvent (e.g., heptane) for analysis byGC.
Several groups of researchers have proposed simplified procedures
for creating themethyl esters. For example, lipids can be trans-
methylated
in situ
. This option combines all of the conventional
steps, except for the drying and post-reactionwork-up, into one
step.
4
For some samples, trimethyl-sulfonium hydroxide (TMSH), an
alternative derivatization reagent, can be used for transesterification.
Amajor advantage of this approach is that the derivatization can be
performed in a single, fast reaction step.
AnalyzingPolyunsaturatedFAMEs
Stabilwax
®
andRtx
®
-Wax columns provide excellent resolution of
FAMEs derived from either plant or animal sources. Polyunsaturated
FAMEs typically are analyzed on one of theseCarbowax
®
-type cap-
illary columns; analysis times of 35-50minutes generally are
required to fully resolve theC21:5 FAME from theC23:0 internal
standard, and theC24:0 FAME fromC22:6.
The FAMEWAX
®
polyethylene glycol stationary phase is specially
testedwith a polyunsaturated FAMEsmix to ensure resolution of
the omega-3 and omega-6 fatty acids of interest, including those
specified above. In addition, FAMEs such asmethyl eicosapaen-
tenoate (C20:5) andmethyl docosahexaenoate (C22:6), found in
nutraceutical ingredients and products such asmarine oils, also are
resolved. FAMEWAX
®
columns offer excellent resolution of
polyunsaturated FAMEswith significantly reduced analysis times,
compared to traditional Carbowax
®
stationary phases. In fact, analy-
sis times of less than 10minutes are possible! Figure 1 shows an
Figure 1—AFAMEWAX
®
capillary columnprovides fast, efficient separations ofmarine oil-basedFAMEs.
Analysis completed in
less than 10minutes!
1. C14:0
2. C14:1
3. C16:0
4. C16:1
5. C18:0
6. C18:1 (oleate)
7. C18:1 (vaccenate)
8. C18:2n6c
9. C18:3n3
10. C20:0
11. C20:1n9
12. C20:2n6
13. C20:4n6
14. C20:3n3
15. C20:5n3
16. C22:0
17. C22:1n9
18. C24:0
19. C22:6n3
20. C24:1
FAMEWAX
™
30m, 0.32mm ID, 0.25µm (cat.# 12498)
Sample:
10mg/mL total FAMEs
Inj.:
0.5µL, split (150:1), 3mm ID split liner
for Trace SeriesGCs, packed
with glasswool (cat.# 20936-202.1)
Inj. temp.:
250°C
Carrier gas:
hydrogen, constant flow
Linear velocity: 62cm/sec.
Oven temp.:
195°C to 240°C@ 5°C/min. (hold 1min.)
Det.:
FID, 250°C
GC_FF00566
