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separation of C21:5n3, C23:0, andC22:4n6 can be used for these
methods. The peakheight of the internal standard,methyl
tricosanoate (C23:0),must be greater than50%of the peak
heights ofmethyl eicosapentaenoate (EPA) and docosahexanoate
(DHA)
1
. In addition,MethodCe 1b-89 specifies thatmethyl
tetracoanoate (C24:0)must be baseline resolved fromDHA
(C22:6n3)
2
.
Pro ezGC
™
software and aFamewax
™
columnwere used to
optimize the analysis ofmenhaden oil. Using theGC conditions
suggested in thesemethods results in baseline resolution of all
critical components, butwith an analysis time ofmore than45
minutes. The computer programwas able todetermine a faster,
more efficient analysis time that still resulted in the required
separations.Aprogramwith a single temperature ramp rate and a
higher column flow rate allowedbaseline separationof all
critical components inonly30minutes. Figure 2 is the actual
optimized analysis ofmenhadenoil. Analysis timewas reduced
by23minutes, a greater than50% savings in analysis time!
CalculatingECLValues forPolyunsaturatedFAMEsUnder
OptimizedConditions
Since there aremanymono-, di-, andpolyunsaturatedFAMEs in
amenhadenoil sample, identificationbyECL is useful. ECLs
are similar to linear temperature-program indices, but are
calculated relative to saturatedFAMEs insteadof hydrocarbons.
Although the elution order did not changewith the optimized
chromatogramofmenhadenoil, there could still be slight
changes in theECLvalues due to changes in retentionof the
individual FAMEs. To ensure the accuracyof the identifications,
the
Pro ezGC
™
software can recalculate theECLvalues from the
actual retention times of the analyses. After entering a few
marker retention times, such as the saturatedFAMEs in the oil,
the program calculatesECLs for all of the components in the
sample. Table II illustrates the retention times andECLvalues
for theFAMEs inmenhadenoil using the optimized conditions.
Summary
Computermodelingon the basis of TRIs is a powerful, accurate,
and effective tool for optimizing the analysis of FAMEs. The
predicted analysis canbe illustrated clearly in the formof a list of
components and retention times or by a simulated chromatogram.
Comparisons between actual retention times ofC4-C24 saturated
FAMEs andpredictedvs. actual chromatograms of cocoa butter
FAMEs demonstrate the precision and accuracy of computer
modeling. In addition, ECL values can be calculated for the
actual column and conditions used.
1
2
3
4
5
6
7
8
9
10 11
12
min.
5
10
1. C14:0
2. C16:0
3. C16:1n7
4. C16:2n4
5. C16:3n4
6. C16:4n1
7. C18:0
8. C18:1n9
9. C18:1n7
10. C18:2n6
11. C18:3n3
12. C18:4n3
13. C20:0
14. C20:1n9
15. C20:1n7
16. C20:2n6
17. C20:3n6
18. C20:4n6
19. C20:3n3
20. C20:4n3
21. C20:5n3
22. C22:0
13
15
14
19
16
18
17
21
24
20
22,23
28
26
29
32
25
30
27
31
15
20
25
30m, 0.25mm ID, 0.25µm Famewax
™
(cat.# 12497).
0.8µL split injection of menhaden oil PUFAwith C23:0 (IS).
On-column concentration 100-150ng.
Oven temp.:
120°C to 220°C@ 7°C/min.
(hold 20min.).
Inj. &det. temp.:
220°C
Carrier gas:
hydrogen
Linear velocity:
60 cm/sec. set@ 120°C
FID sensitivity:
8 x 10
-11
AFS
Split ratio:
50:1
23. C22:1n9
24. C22:2n6
25. C21:5n3
26. C23:0
27. C22:4n6
28. C22:5n6
29. C22:5n3
30. C24:0
31. C22:6n3
32. C24:1n9
Figure2
MenhadenOil PUFAAnalysis (Actual Chromatogram)
References
1. Association of Analytical Chemists, International AOACOfficial
Methods of Analysis. 15th Ed.: 3rd Supplement, 1992, pp 140-142.
2. American Oil Chemists Society, Official Methods and Recommended
Practices of the American Oil Chemists Society, 1994. 8.
3. Christie, W.W., Gas Chromatography and Lipids, 1989, pp 92-96.
