Biology Reference
In-Depth Information
FIGURE 13.10
Diagram
of a gas chromatograph
[29]
.
Sample
injector
Flow controller
Waste
Column
Detector
Carrier gas
Column oven
glycol, H-(OCH
2
CH
2
)
n
-OH)). The analytes partition to a different extent between the mobile
gas phase and the stationary phase. Analytes that favor the gas phase leave the column faster
and so have shorter retention times. The column is located in an oven to assure accurate,
programmable temperature control. It is an elevated temperature that volatizes the fatty
acid methyl esters and makes GC feasible. The fatty acids must be methylated to make
them volatile. The final components of a functional GC, depicted in
Figure 13.10 [29]
, are
an injection port and a detector. There are many types of detectors used in GC, the most
common being the flame ionization detector (FID) and the thermal conductivity detector
(TCD).
Free fatty acids, as well as fatty acids esterified to other biochemicals (e.g. acyl glycerols,
cholesterol esters, phospholipids), are not volatile and if heated to a high enough tempera-
ture would disintegrate. However, methyl esters of fatty acids are volatile and can be used
in GC analysis
[30]
. Preparation of fatty acyl methyl esters is perhaps the most commonly
used reaction in lipid analysis. Fatty acid methyl esters can be made from free fatty acids
that have been hydrolyzed (saponified) from their biological ester source (see Chapter 5)
[31]
. However, it is not necessary to actually obtain free fatty acids
before
methylation. Fatty
acid methyl esters can be directly obtained from biological esters by a process known as
trans-esterification
[32]
. Amethod that can trans-esterify can also directly methylate free fatty
acids. Methylation can be achieved in either acidic (by methanolic hydrogen chloride or
boron trifluoride in methanol) or basic (sodium methoxide in methanol) anhydrous condi-
tions. An emphasis must be placed on
anhydrous
as even traces of water can be detrimental
to esterification. The methoxide method is extremely fast (complete in a few minutes) and is
accomplished at room temperature
[33]
.
Figure 13.11
shows a fatty acid profile of liver phospholipids determined by GC
[34]
. Each
fatty acid is identified by three numbers: number of carbons, number of double bonds, and
the series (omega-family). Elution time is on the abscissa and the detector intensity on the
ordinate. The area under each curve represents the relative amount of each fatty acid in
the sample. A fatty acid methyl ester database has been established
[35]
. It contains retention
times and mass spectral information on ~100 fatty acids.
