A note on the use of gas chromatography in biology.

1. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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Gas Chromatography
Gas chromatography is a chromatographic technique that can separate and analyze
volatile compounds in the gas phase.
Depending on the stationary phase used in this analytical technique, there are two
types of gas chromatography: 1. Gas-liquid chromatography (GLC) and 2. Gas-solid
chromatography (GSC). Among these, GLC is the most widely used method. A gas
chromatography looks like:
Figure: Gas Chromatography.
PRINCIPLE OF GAS CHROMATOGRAPHY
All chromatography techniques have one stationary and one mobile phase. In this
chromatography, the mobile phase is always gas. But the stationary phase is either
liquid or solid. If the stationary phase is solid, then that is called gas-solid
chromatography or GSC. And if the stationary phase is liquid, then that is called gas-
liquid chromatography or GLC. In GLC, the mobile gas phase is like helium and the
stationary phase is a high boiling point liquid adsorbed onto a solid. As in other
chromatographic techniques, the mobile phase is a chemically inert gas which carries
the analyte through a heated column to separate into its compounds.
WORKING / COMPONENTS OF GAS CHROMATOGRAPHY

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Among various types of gas chromatography, GLC or gas-liquid chromatography is
the most popular one. This chromatography consists of an injection port, a column, an
oven, a heater to control the temperature, a carrier gas flow control equipment, and a
detector.
Injection Port: An analyte (the substance to be analyzed) in a very small quantity is
injected into the machine through a rubber septum at the injection port using a small
syringe. The sample is then heated at 50oC, above the boiling point of the sample for
vaporization. The sample vapor is then carried by a mobile gas phase helium into the
column.
Column: There are two types of columns in gas chromatography: one is a long thin
tube packed with a stationary phase and the other is even thinner where the stationary
phase is bonded to the inner surface of the column. A packed column is normally made
of stainless steel and coiled up so that it can easily fit inside an oven. The column is
1-4 meters long with a diameter of up to 4 mm. The column is packed with
diatomaceous earth, a very porous rock. This finely grounded solid is coated with a
high boiling liquid-like waxy polymer. The separation in the column depends on three
things:
 The components in the mixture can be carried along by the mobile phase, helium
gas. It depends on the boiling point of certain components in the mixture and the
temperature of the oven. If the boiling point is lower than the temperature of the
oven the components will move along with the helium gas.
 The components can be dissolved in the liquid stationary phase. The more soluble
components will spend more time in the liquid phase and less soluble components
will spend less time in the liquid phase. The less soluble compounds will tend to
leave the liquid phase and move along with the gas phase.
 Some components can condense on the stationary phase. If the boiling point of
some components is higher than the temperature of the oven, they may condense
on the stationary phase. But it will eventually move on due to the long time

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exposure of heat like the water on the surface of the sea evaporates slowly on a
hot day.
This way the molecules spend some time in the stationary phase and sometimes move
along with the mobile phase to reach the detector.
Oven: The column stays inside a thermostatically controlled oven where temperature
can be varied between 50-250oC. This oven doesn't allow heat transfer from or to the
column. The temperature of the injection oven is higher than the column oven. The
high boiling components can be condensed at the beginning of the column. But as the
analysis proceeds the temperature of the column oven rises which can be controlled
if necessary.
Detector: A flame ionization detector is most commonly used in Gas chromatography.
In this type of detector organic molecules are burned by flame to get ions and
electrons. The positive ions are attracted to the cathode, while the negative ions are
attracted to the anode. So, the electrons flow from electron-rich anode to electron-
deficient cathode through the external circuit. This electric current is more if we have
more organic compounds coming out of the column and it is less if there are fewer
compounds. Depending on the amount of current passing through the circuit the
computer plots a graph and we can see that on display.
APPLICATIONS OF GAS CHROMATOGRAPHY
Some applications of gas chromatography are as follows:
Qualitative Analysis: Here the compounds in a given sample are identified. Qualitative
analysis is done by measuring and comparing the retention time of the sample with
that of the standard. The retention time for the sample and standard would be the
same under identical conditions.
Testing The Purity Of Compounds: When the chromatogram is run, the peaks are
checked. The presence of additional peaks is an indication of impure compounds. For

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this, the chromatogram of the test is compared with that of the standard. Based on the
percentage area of the peaks, the percentage of purity can be derived.
To Trace Impurities: This is similar to the previous step. The additional peaks indicate
the presence of impurities. The peak area also helps to determine the percentage of
impurities in the test sample.
Quantitative Analysis: This is the technique that helps to determine the quantity of the
compound in the test sample. This is done by either: (a) direct comparison technique,
(b) by use of calibration curves, and (c) by internal standard method. In general,
substances that vaporize below 300°C (and therefore are stable up to that
temperature) can be measured quantitatively. The samples are also required to be
salt-free; they should not contain ions. Very minute amounts of a substance can be
measured, but it is often required that the sample must be measured in comparison to
a sample containing the pure, suspected substance known as a reference standard.
Various temperature programs can be used to make the readings more meaningful;
for example to differentiate between substances that behave similarly during the gas
chromatography process.
Determination Of A Mixture Of Components: This is a multicomponent analysis
wherein the quantity of each component in the mixture is determined. This procedure
is mostly used for quality control of pharmaceutical drugs. Professionals working with
GC analyze the content of a chemical product, for example in assuring the quality of
products in the chemical industry; or measuring toxic substances in soil, air, or water.
GC is very accurate if used properly and can measure pico-moles of a substance in a
1 ml liquid sample, or parts-per-billion concentrations in gaseous samples.
Isolation And Identification Of Metabolites: The biological samples like urine, serum,
plasma can be analyzed to identify the metabolites in them. In practical courses at
colleges, students sometimes get acquainted with the GC by studying the contents of
Lavender oil or measuring the ethylene that is secreted by Nicotiana benthamiana

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plants after artificially injuring their leaves. This GC analyses hydrocarbons (C2-C40+).
In a typical experiment, a packed column is used to separate the light gases, which
are then detected with a TCD.
Identification Of Compound Mixtures: A mixture of compounds like amino acids,
volatile oils, plant extracts can be identified by the use of gas chromatography. Some
of the pharmacopeias like USP, BP recommend gas chromatography analysis for
drugs like diphenhydramine, atropine, antazoline, etc. This is a standard protocol to
be followed by industries as part of quality control.
Forensics: Gas Chromatography is used extensively in forensic science. Disciplines
as diverse as solid drug dose (pre-consumption form) identification and quantification,
arson investigation, paint chip analysis, and toxicology cases, employ GC to identify
and quantify various biological specimens and crime-scene evidence.