Gas chromatographic analysis of organic metabolites has been used widely for the analysis of fats,
fatty acids, pesticides and several volatile substances. Extensive studies have been made possible
on organic volatiles in food and fermented beverages where flavour and aroma are of utmost
importance. The technique was first developed in 1941 and extended to the estimation of nitrogen
fixation in 1966 using the reduction of acetylene to ethylene.
This discovery has greatly expedited subsequent research on, and analysis of, N,-fixation.
Formerly, nitrogen fixation was carried out using the heavy isotope N" and mass spectrometry.
Of course, since only two electrons are required to reduce a molecule of acetylene to ethylene,
whereas the reduction of a molecule of N, to NH, requires six electrons, acetylene reduction data
must be divided by a factor of 3 when converting to equivalent N,-fixation.
In principle, the compounds in a mixture migrate at different specds through a tube packed
with a solid adsorbent by adsorption partitioning. The fractionating column of a typical gas
chromatogram consists of a copper or stainless steel tube (about 1/4m in diameter and 1-4 m long)
packed with an inert material such as firebrick or diatomaceous earth that has been pulverized and
coated with a nonvolatile liquid called a partitioner. After the tube has been packed, it is normally
bent into a series of U-turns or wound into a helix so that it can be fitted easily into an insulated
heated box. As the sample is moved through the column by the carrier gas (helium, nitrogen, etc.),
the partitioner must interfere in a selective fashion with the progress of each compound present
slowing up the progress of some and permitting others to travel through the column more swiftly.
The difference in the partition coefficients of various components in the mixture is the primary
basis for separation. At the outlet of the column, a detecting device signals the emergence of each
compound by activating a pen on a strip-chart recorder. The job of the detector is not to identify
the emerging compounds, but to signal the presence or absence of foreign particles in the emerging
gas. The emerging material is actually detected by a thermal conductivity cell (particularly for fatty
acids) or a hydrogen flame analyzer.
As mentioned earlier, GLC is extremely helpful in analyzing mixtures of fatty acids of different
chain lengths. However, before the fatty acids are analyzed by chromatography, they must be
separated from esterifed compounds. This is done by saponification reaction which breaks the ester
bonds and releases the fatty acids. Following saponification, the carboxyl group must be esterified
again with a methyl group of prevent it from reacting in the chromatography tube. GLC has also
been useful for the analysis of steroids, amino acids and hydrocarbons.
