2. Introduction
• Gas chromatography (GC) is an analytical technique that helps to separate & analyze a mixture of organic
vapourizable or volatile compounds without their decomposition.
• It is carried out at a suitable temperature in a glass or metal tubing known as the column, which contains
the liquid or solid stationary phase.
• Inert gases like helium or unreactive gases like nitrogen are used as mobile phase which are passed over the
stationary phase.
• The instrument used to perform gas chromatography is known as gas chromatogram or gas separator or
aerograph. The technique for gas chromatography is similar to that of column chromatography except that
the liquid mobile phase in the latter is replaced by a moving gas.
• Gas chromatography is frequently used term to represent gas-liquid chromatography that employs the
principle of partition.
Types of Gas Chromatography:
1. Gas-Liquid Chromatography
2. Gas-Solid Chromatography
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3. Principle
The principle of gas chromatography is similar to that of column chromatography, HPLC as well as TLC with
the following differences,
1. In the gas chromatography, the separation of mixture of components occurs between a gaseous mobile
phase & a liquid stationary phase whereas in other chromatographic techniques, it occurs between a liquid
mobile phase & a solid stationary phase.
2. The concentration of solute (sample) components in the carrier gas is entirely a function of vapour pressure
of the carrier gas.
3. In gas chromatography, the column is placed in an oven whose temperature can be controlled whereas in
other chromatographic techniques, temperature programming is not essential.
Gas chromatography generally employs an injection port, a carrier gas as mobile phase, a column as
stationary phase, a detector & a data recording system.
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4. A known volume of sample to be analyzed is taken in a microsyringe & injected into the injection port
maintained at a temperature higher than the boiling point of the sample.
The components of the sample gets vapourized.
When a continuous carrier gas (mobile phase) is passed through the injection port, it sweeps the vapourized
sample onto the column (stationary phase).
The components of the sample get distributed between the two phases.
Since the absorption or solubility properties of the components of the sample differ, the components are
carried along the column at different rates and at different retention times.
The separated components emerge from the column in distinct peaks which is then detected by a suitable
detector supplied with an automatic data recording system.
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5. Theory
The concept of gas chromatography is dependant upon the following parameters.
1. Retention Time (Rt)
◦ It may be defined as the absolute time taken by a sample to show maximum peak on the gas chromatogram
after injecting.
◦ It may be considered as the total time spent by the sample in the stationary phase and the mobile phase.
◦ It is completely dependant upon the operational conditions & is expressed in minutes.
2. Retention Volume (VR)
◦ It may be defined as the volume of gas required to elute about half of the solute through the column.
◦ It is the product of retention time (tR) & the gas flow rate (f).
VR= Rt x f
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6. Instrumentation
◦ Modern gas chromatography utilizes a complex instrument that usually consists of four units supported by
three temperature controllers (ovens).
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1. Gas Supply Unit
• Carrier gas supply unit includes a large gas tank or a cylinder filled with compressed gas that acts as mobile
phase.
• The cylinder is fitted with a pressure controller to control the pressure of gas, a pressure gauge that
indicates the pressure, a molecular sieve or a filter drier to transfer filtered dry gas & a flow regulator to
ensure a constant rate of flow of mobile phase to the column.
Carrier Gas:
◦ The main function of carrier gas is to move or sweep the vapourized sample towards the column. It should
meet the following criteria;
1) It should not react with the column or sample i.e., it should be chemically inert.
2) It should be cheap & readily available.
3) It should be of high quality (purity) & should not cause any fire accidents.
4) It should give best possible results.
5) It should be suitable for the sample to be analyzed & for the detector.
◦ Hydrogen, helium, nitrogen & carbon dioxide are the gases used in GC.
◦ Hydrogen has low density & better thermal conductivity. However, it reacts with unsaturated compounds &
is inflammable & explosive in nature.
8. 2. Sampling Unit
• Sampling unit or injection port is a piece of hardware attached to the column head.
• It provides a means for the introduction of solute (sample) into the carrier gas.
• The carrier gas that continuously flows from the cylinder, enters the injection port & sweeps the vapourized
sample towards the column.
• Since the sample to be analyzed should be in vapourized state (a gas), the injection port is provided with an
oven that helps to maintain its temperature at about 20-50°C above the boiling point of the sample.
• The sample to be analyzed may be in gaseous , liquid or even in solid state.
• Gaseous samples may be most conveniently introduced into the injection port by the use of gas tight
hypodermic needle of 0.5-10 ml capacity.
• For liquid samples, microsyringe of 0.1-100 µL capacity may be used.
• Solid samples may be dissolved in suitable volatile solvents before injection or may be packed in fragile gas
ampoules, which are then introduced into the injection port provided with small heating coils & are quickly
crushed.
• The sample then instantaneously gets vapourized & flows into the column under the influence of the carrier
gas.
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9. Injectors in GC:
1) Cold Injector-
a) On-Column Injector (OCI)-
It uses a special type of syringe with a needle that is narrow at the tip & with a column of 0.53 mm internal
diameter. The sample is transferred directly to the column without splitting. It allows programming of the
injection temperature.
2) Vaporization Injector-
a) Direct/ Wide Bore Injector-
In this the sample is introduced by splitting the flow of the carrier gas to column & purge vent where almost
the entire sample is injected.
Like on-column injector it uses a column of 0.53 mm I.D. when required & the peaks obtained are broader
when compared to those obtained by split injector.
b) Split/Splitless Injector-
It is the most common type of injector used in GC & is suitable for narrow as well as wide bore capillary
columns.
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10. 3. Column Unit
Mixture of vapourized sample & the carrier gas from the injector port enter the column, that serves as the
heart of the gas chromatogram.
The different components of the vapourized sample get separated from each other depending upon their
different interactions with the packing material of the column. The separated components then enter the
detector.
Columns of different shapes & sizes may be used. The commonly used shapes include ‘U’ tube type & the
coiled helix type wherein the former is easy to pack & is advantageous due to its shorter length, while the
latter serves as the best possible shape but is difficult to be packed evenly.
The chromatographic columns are made-up of copper, stainless steel, glass, aluminium, nylon & several
other synthetic plastics.
Of all these materials, glass is most commonly used.
The columns are filled with support material as well as liquid phase (stationary phase).
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11. a) Support Material:
• The function of the solid phase in GC is to provide mechanical support to the liquid phase. An ideal support
should have the following properties;
i. It should be a poor adsorbent.
ii. It should have a large surface area.
iii. It should be chemically inert.
iv. It should get uniformly wet with the liquid phase.
v. It should be thermostable.
vi. It should be strong enough to prevent the fractionating of the column.
• The commonly used solid phases include diatomaceous earth or kieselguhr, firebrick , glass beads,
unglazed tiles, porous polymers, sand etc.
b) Liquid Phase:
• Many liquid phases are available for the preparation of columns for gas chromatography.
• However, the choice of the suitable liquid phase depends upon experience or trial & error.
i. It should be non-volatile or have low volatility i.e., should be stable at the operating temperature.
ii. Should have high decomposition temperatures.
iii. Should be chemically inert.
iv. Should exhibit less viscosity at operating temperature.
v. Should possess low vapour pressure at column temperature. 11
12. • For good results, the liquid phase should be chemically and structurally similar to the solute (sample) i.e.,
polar liquid phase for polar solute & non-polar liquid phase for non-polar solute.
c) Types of Columns:
1. Packed Columns-
o Packed columns are prepared from glass or metals. They are 2-3 m long with an internal diameter of 2-4
mm.
o In GLC, these columns are densely packed with finely divided solid support which is in turn coated with a
thin layer of liquid stationary phase.
o In GLC, the columns are packed with adsorbents or porous polymers. Packed columns are shaped as coils.
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2. Capillary Column/Open Tubular Columns-
o Capillary columns contain simple capillary tubes made up of glass, metal or quartz.
o Their lengths ranges from 10-100 m & their inner/diameter is usually 0.1-0.5 mm.
o Support-coated open tubular (SCOT) columns are a type of capillary columns.
o Their inner walls are coated with a thin film of supportive material like diatomaceous earth onto which the
stationary phase is adsorbed.
o SCOT columns are also known as porous-layer open tubular (PLOT) columns.
o Capillary columns provide the highest efficiencies & greatest resolutions in lesser time.
d) Equilibration of Column:
• The packed columns are equilibrated before the introduction of the sample.
• This is done by allowing continuous flow of heated carrier gas through the columns for a specific duration of
time at the prescribed temperature.
14. 4. Detectors
1) Thermal Conductivity Detector (TCD)/ Katharometer Detector:
Principle-
TCD is based upon the fact that the heat lost from a filament depends upon the thermal conductivity of the
stream of the surrounding gas as well as its specific heat.
It detects the changes occurring in the thermal conductivity of an eluent from the column & compares it
with the mobile carrier gas.
Thermal conductivity of most of the compound is lesser than the commonly used carrier gases.
Construction & Working-
TCD consists of a temperature controlled metal block into which two cylindrical chambers or cavities of
small volumes are made.
Both the cavities are supplied with filaments made up of platinum, tungsten or alloy. The filaments have
high temp. coefficients of resistance & constitute the reference (R) & the sensing (S) elements.
Both these filaments are placed in the arms of the Wheatstone bridge arrangement & electrically heated.
At a given temperature when carrier gas alone is allowed to flow through the sensing cell.
The temperature of the filament in S changes. This is because the thermal conductivities of both, the sample
& the carrier gas are different.
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15. Such difference results in a change in the heat loss from the filament ultimately leading to a change in the
resistance of S.
Such resistance changes are sensed by the Wheatstone bridge arrangement. The bridge then produces a
measurable voltage change that is amplified & signaled to the recorder.
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2) Electron Capture Detector (ECD):
Principle-
Molecules of compound, which possess affinity for electrons, differ in their electron absorbing capacities . This
difference is utilized in the electron capture detector for identification of compounds.
Construction & Working-
A foil made up of a radioactive metal like Ni 63 or titanium tritide is placed inside a teflon coated cell which
also contains a cathode & an anode for the purpose of electron repulsion & electron capture respectively.
The cell has an inlet for allowing the entry of eluted sample along with carrier gas an outlet for the exit of the
components.
The electron emitted from the radioactive source have sufficient energy to excite the atoms of carrier gas
which in turn help in ionizing the eluted sample.
The current produced as a result of ionization of eluent is recorded in the form of peaks & this helps in
identification of the components.
18. 3) Flame Ionization Detector (FID):
Principle-
• When organic compounds are introduced into a hydrogen-oxygen or hydrogen-air flame they get ionized thus
producing current.
• Carrier gas produces low levels of current or no current at all. Therefore, current produced from the sample
ionization is detected after its amplification.
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Construction & Working-
• A typical FID employs hydrogen as a combustion gas. A tiny flame of hydrogen is maintained in a small
cylindrical jet made up of platinum or quartz.
• Air or oxygen is introduced into the jet which aids in combustion.
• Effluent from the column with helium or nitrogen as carrier gas are fed into the hydrogen flame.
• The effluent mixes with the hydrogen fuel and oxidant, gets ignited and undergoes pyrolysis to produce ions.
• For the detection of these ions two electrodes (+ve and –ve) are used that provide a potential difference.
• The ions produced are repelled by the positive electrode which hit the collector plate.
• The current produced in doing so is amplified & fed to an appropriate recorder.
• Any change in the combustion of elute from the column changes the current flow.
20. Working of Gas Chromatogram
1. Take a suitable syringe & wash it 2-3 times with acetone. Fill it carefully with the sample.
2. Switch on the recorder & set it at an appropriate speed.
3. Record the setting i.e., column temp, detector temp. and injection port temp.
4. Introduce about 1-2 µl of the sample into the injection port by completely inserting the needle into the
rubber septum. This step is to be carried out quickly because the temperature of the injection port is kept
very high. Note down the injection time.
5. The sample gets vapourized due to the higher temperature of the injection port & is swept towards the
column by the carrier gas.
6. The vapourized sample components now get distributed between the gas and the liquid phase and then get
solubilized in the liquid stationary phase depending upon their solubilizing tendencies.
7. Hence, rate of movement of sample components along the column depends upon their tendencies to get
solubilized in the liquid phase i.e., the component that have minimal solubility move faster through the
column while those with maximum solubility travels slowly.
8. The components leaving the column activate the detector and recorder to give a plot. Generally
potentiometric recorder are used for recording the signals from the detector.
9. The components that slowly leave the column give a bell shaped curve of shorter peak while the one which
travels faster give a bluntly pointed curve of larger peak. Moreover, the area under the individual peaks is
proportional to the concentration of the compound responsible for that particular peak.
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22. Derivatization Techniques
◦ GC is considered to be the best possible technique for the separation of volatile compounds that remain
thermally stable during the process.
◦ However, certain functional groups like NH2, OH, NH, SH & COOH do not fulfill this criteria & present
problems in GC separation. Hence they need to be derivatized.
A. Silylation:
• It is the most commnly practiced derivatization technique in GC and widely used group for this purpose is
trimethylsilyl (TMS) or [Si(CH3)3].
• Derivatization by silylation involves the conversion of polar NH2, OH, SH and COOH groups into non
polar, more volatile and thermally stable group.
• The silyl group upon reaction with amines, amides, alcohols, acids and thiols undergo replacement with
their active hydrogen resulting in the formation of silyl derivatives.
• A large number of silylation reagents are available for this purpose. Most of these reagents and their
derivatives have good thermal stability and are compatible with a vast range of injection port and column
conditions.
• Both silylation reagents as well as their derivatives undergo decomposition in the presence of moisture.
Hence, they should be protected from moist environment.
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2. Alkylation:
• Derivatization in GC by alkylation reaction involves the replacement of an active hydrogen in R-OH, R-SH, R-
NH2 or R-COOH with an alkyl group. This results in the conversion of organic acids into esters, especially
methyl esters.
• The technique is commonly employed in GC because the chromatogram produced by esters is better than that
by organic acids.
• Moreover, alkylation derivatives are less polar, highly stable & can be separated & stored for longer durations
than the parent compound.
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3. Acylation:
• This derivatization technique includes the conversion of compounds with active hydrogens by the action of a
carboxylic acid or its derivatives.
• E.g: OH to esters, NH to amides, SH to thioesters.
• The derivatives so formed are better chromatographed & detected than the parent compound.
• Acylation may be carried out by following two types of acylation reagents, fluoro acid anhydride &
fluoroacylimidazoles.
25. Temperature Programming
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Column temperature is an important variable which for better results should be controlled to few tenths of a
degree.
The optimum temperature of the column depends upon the boiling point of the sample as well as on the
degree of separation.
The temperature of the column can be controlled by jackets equipped with vapors of a boiling liquid,
electrically heated metal blocks or circulating air baths. The technique used to control the temperature of
columns is known as temperature programming.
At high temperatures, the rate of elution increases while the adsorption power decreases.
Therefore most of the separations are carried out at a room temperature.
Low temperature favors increased recovery of sample while high temperatures are used for separating
difficult samples.
26. Applications of GC
Qualitative Applications
Quantitative Applications
Calibration curve method
Direct comparison method
Internal standard method
Detection of impurities
Detection of mixture of drugs
To check purity of compound
Isolation & identification of drugs
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