This document provides information about gas chromatography. It discusses the key components of a gas chromatography system including the mobile phase, stationary phase, columns, temperature control, sample injection systems, and various detectors. The mobile phase is usually an inert gas like helium, hydrogen, or nitrogen. Stationary phases can be solid adsorbents or liquid coatings. Columns include packed columns and capillary columns. Temperature control programs are used to separate compounds of varying boiling points. Common detectors mentioned are the thermal conductivity detector, flame ionization detector, and electron capture detector.

1. Mr. Sanket P. Shinde
Assistant Professor
Pune-Maharashtra.

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Introduction
• Gas chromatography is one of the widely used chromatographic
techniques, which use inert gas as the mobile phase.
• In gas chromatography the components of a sample, after vaporization,
are separated by being partitioned between gaseous mobile phase and
solid or liquid stationary phase.
• The inert gas does not interfere with the analyte but transport the
components through the column and facilitate the separation.

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Theory & Principle
• Gas chromatography requires a mobile phase and a stationary phase like all
other chromatography techniques.
• The mobile phase is comprised of an inert gas such as helium, argon or
nitrogen.
• The stationary phase consists of a packed column in which the packing or
solid support, or a liquid coat act as stationary phase.
• The main principles involved are adsorption and partition for gas solid
chromatography and gas liquid chromatography, respectively.
• In gas solid chromatography the analytes of the mixture distribute
themselves between the gas phase and the adsorbent.

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• The difference in the adsorptive behavior causes separation, whereas in gas
liquid chromatography, the partition co-efficient is the main factor for the
separation.
• The analytes of mixture distributes themselves between gas phase and
liquid phase according to their partition co-efficient.
• The separation of compounds is based on the different strengths of
interaction of the compounds with the stationary phase.
• The stronger the interaction is, the longer the compound interacts with the
stationary phase, and the more time it takes to migrate through the column
thus lead to longer retention time.
• The separation of the analytes depends upon the vapour pressure of the
compound, polarity of the compound and stationary phase, column
temperature and flow rate of mobile phase.
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Phases used in Gas Chromatography
1. Mobile Phase (Carrier Gas):
• The mobile phase involved in the gas chromatography is gas.
• As the gas is used to transport the sample components through the column,
it is also called as carrier gas.
• Carrier gases are compressible gases that expand with increasing
temperature.
• This results in a change in the gas viscosity.
• The selection and linear velocity of the carrier gas will affect resolution and
retention times.
• The commonly used carrier gases are hydrogen, helium, argon and nitrogen.
Each gas has its own advantages and disadvantages.

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An ideal carrier gas should meet the following requirements
1. The carrier gas should be inert, so that it will not react with the stationary
phase, sample to be analyzed and thus will not interfere with the separation.
2. It should be comfortable with the detector.
3. It should be easily available and not much expensive.
4. It should be highly pure (99.9%).
5. It should be easy for handling in terms of fire hazard.
6. It should contribute minimally to the partitioning process.

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Hydrogen
This is mostly used as carrier gas. It has better thermal conductivity and low
density thus suitable for thermal conductivity detector and Flame ionization
detector. It has the lowest viscosity at any temperature, so it will produce
higher velocities at a given pressure drop.
Helium
It has got a excellent thermal conductivity and low density. It is inert gas and
allows greater flow rate, suitable with thermal conductivity detector. It is
preferred than hydrogen due to its safe use. However it is expensive.
Nitrogen
This is inexpensive compared to helium and used in flame ionization detector.
But the limitation is the reduced sensitivity.
Argon
It is most suitable for electron capture detector but it is not easily available.
Carrier Gas

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• Carrier gases are usually stored in cylinders in compressed form.
• Another option is the gas generators that produce gases continuously.
• The filters, drier and adsorbing cutes are used to remove the moisture
and offer gas contaminants to ensure the purity.
• The gas flow rate in cylinders is controlled by pressure regulators that
are attached with pressure gauge.
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2. Stationary Phases
• Gas Solid Chromatography: In gas solid chromatography, stationary
phases are solid adsorbents. The analyte is adsorbed on the surface of solid
adsorbents. Different types of adsorbents are used as stationary phases.
• Gas Liquid Chromatography: In gas liquid chromatography stationary
phases are liquids supported by solid. The liquid phase is coated over a
solid support and the analytes distributed between gas and liquid.

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Types of Gas Chromatography
1. Gas Liquid Chromatography (GLC)
• The principle involved in gas liquid chromatography (GLC) is partition.
• The components of the sample to be analysed is partitioned between the
inert gas, (mobile phase) and a liquid phase which is coated on a solid
support or the wall of a capillary tube.
2. Gas Solid Chromatography (GSC)
• In this technique the stationary phase is solid adsorbent.
• The principle involved in gas solid chromatography is adsorption.
• The separation of components of sample is achieved by physical
adsorption of components thereby retention on the solid stationary phase.
• This technique is not widely used.

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Instrumentation

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• This consists of gas cylinders and generators to produce carrier gases.
• Read the pressure of gas and flow rate of the carrier gases is controlled by
the pressure regulators.
• Some of the contaminates and the methods to remove them are given
below.
a. Air or Oxygen
These can oxidize sample components and liquid stationary phases.
This can be removed by a cartridge containing molecular sieve.
b. Hydrocarbons
A cartridge containing activated carbon can be used to remove this
Hydrocarbon.
c. Water Vapour
Water vapour can affect some solid and bonded liquid stationary phases
and performance of some detectors. It can be removed by molecular sieve.
1. Gas Source and Regulation System

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• There are different methods of sample injection system and design of
injection port available.
• The selections of method are depending on the nature of sample and
column to be used.
• Now-a-days automatic samplers are available which function automatically
to inject samples precisely than manual practices.
• Gaseous samples are introduced through a special gas sampling valve or a
gas syringe.
a) Split Injection System
• This is used with capillary columns, to prevent over loading the column
with sample.
• It consists of a hollow needle which is placed centrally that permits only a
small part of the sample to columns.
• The remaining part of sample is allowed to the atmosphere by a control
valve.
2. Sample Injection System

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b) Splitless Injection System
• This is used when the sample contains very low level of some components
to be analyzed.
• When the sample is injected control valve is kept closed.
• The top of the column is cooled to just above the boiling point of the
solvent.
• So, solvent travels down the column and sample components are escaped.
• Then the control valve is opened to purge any sample vapours left out,
column temperature is raised and the solutes are released into the gas stream.

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c) Special Injection Techniques
• This includes head space sampling, purge and trap and pyrolysis.
• Head space sampling is useful for the analysis of gases and low viscosity
liquids specially samples with volatile organic constituents such as wine,
environmental samples, food products, blood and water.
• Purge and trap technique is also can be used for volatile organic constituent
and volatile metabolite in wine samples.
• Pyrolysis involved the thermal fragmentation. In this system a
ferromagnetic wire is heated to its curie point, which gives a pyrolysis
temperature.
• This causes the volatile fragments and is introduced into the column for
analysis. This is useful for analysis of low volatile compounds such as
rubber, polymer, paint films resins, microorganism's soils.

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3. Column
• Generally gas chromatography columns are made up of glass or metal
tubing.
• Two types of columns are in general use which are further divided into sub
types as given below.
A. Packed columns
B. Capillary columns
i. Packed column with solid particles
ii. Open tubular column
a) Wall coated open tubular columns (WCOT)
b) Supported coated open tubular columns (SCOT)
c) Porous layer open tubular columns (PCOT)

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A. Packed Columns
• These columns are used for both gas solid chromatography and gas liquid
chromatography.
• The granular stationary phase is packed in glass, stainless steel or nickel
tubing to produce packed columns.
• The length is often 3 meters and inner diameter may range from 1.6 to
9.5mm for gas liquid chromatography columns.
• They are packed with an inert support, usually a diatomaceous earth.
• For non-polar liquids, the support must be inactivated when polar
compounds are to be analysed.
• The surface mineral impurities are removed by acid washing and will be
more suitable for non polar sample analysis.
• For GSC, the columns are packed with absorbents such as silica gel a
bonded phase support, a molecular sieve or porous polymers.

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B. Capillary Columns
• These columns are usually made up of fused silica which is a very high
purity glass.
• The silica tubing has a high tensile strength thus the columns are made as
thin walled and more flexible.
• The outer wall of the column is coated with polyimide to protect the wall
from scratches.
• These columns are further divided into two main types.
i. Packed columns with solid particles
• These columns are also called as micropacked columns. These columns are
packed with solid particles over the whole diameter of the column.

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ii. Open Tubular Column
• The open columns are made up of stainless steel, copper, nylon or glass.
• It is constructed with an open and unrestricted flow path through the
middle of the column.
These columns are divided into following subtypes.
(a) Wall coated open tubular columns (WCOT)
(b) Supported coated open tubular columns (SCOT)
(c) Porous layer open tubular columns (PCOT)
• The whole principle for the above three types of columns are same. They
differ only in some aspects such as length and diameter.
• In these columns the inner wall is coated with the stationary liquid phase.
• The uniform coating of surface was maintained by the introduction of
polymers that wets the surface and produces a uniform film.
• The advantages of WCOT columns over packed columns are rapid
analysis, thus shorter retention times, inertness, longer life's bleed, higher
efficiency and reproducibility.

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• The temperature program controls regulate the increase of temperature during
the analysis.
• In GC, separation of components differing widely in their boiling point.
• In this system, a lower temperature is selected initially. Then the temperature is
increased to push out the higher boiling point components.
• The temperature is increased immediately after sample injection and kept
constant at the program level until the high boiling components are eluted out.
Then the temperature is returned to normal.
• The initial temperature is maintained for 10 minute and then temperature is
increased.
• The third method involves to increase the temperature step by step to reach the
final temperature.
• In linear temperature GC requires a dual column system to compensate the
column bleeding. Separate heaters are needed for heating injection, column
oven and detector systems.
3. Temperature Control Program

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4. Detector
Different types of detectors are used in gas chromatography which are given
below
A. Thermal Conductivity Detector (TDC)
B. Flame Ionisation Detector (FID)
C. Electron Capture Detector (ECD)
D. Flame Photometric Detector
E. Electrolytic Conductivity Detector
F. Other Detector

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• This is also called as differential thermal conductivity detector.
• The principle involved in the detector is based on the thermal conductivity
of a gas which controls the temperature thus resistance of a wire.
Construction
• A tightly coiled filament made up of tungsten/tungsten rhenium tungsten
alloy sheathed with gold is placed inside the cavity of a metal block.
• A DC supply provision is there to heat the filament.
• The standard detector consists of four filaments placed within one metal
block.
• Further a TCD can be constructed with thermistor which is a metal oxide
lead with electrical load attached

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Circuit diagram of Thermal Conductivity Detector

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Working
• The filament is heated by a DC supply to a constant temperature, but less
than a dull red condition when only carrier gas is flowing through the
detector.
• The heat loss from the filament to the metal block is constant.
• When sample component enter into the detector the temperature of the
filament changes.
• Usually the presence of organic materials causes a relatively large decrease
in the thermal conductivity of the column effluent especially with
hydrogen or helium is used as gas.
• The heat loss from the filament decreases, the temperature rises and the
resistance increases.

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• In standard detector, one pair of filament receives the column effluence and
other pair receives the carrier gas.
• The imbalance between these two pairs is recorded.
• Initially only carriers gas is passed through both pairs and a line is
established and then the power supply is adjusted.
• In TCD with thermistor one bead is placed in the pure carrier gas stream
and the other is placed for the column effluents.
• Thermistors are used to work at ambient or sub ambient column
temperature.
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• The principle involved in this detector is electrical conductivity of gases.
• If electrons are present in a gas stream it becomes conductive.
• The change of conductivity is recorded in terms of electric current.
Construction
• It consists of two electrodes, a flame jet and a collector electrode.
• The flame jet is a capillary jet that can produce a flame.
• The collector electrode is placed just above the tip of the flame.
• There are inlets for hydrogen gas, air and column effluent.

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Flame Ionisation Detector

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Working
• When column effluents are allowed to enter into the FID, Hydrogen gas is
passed through the inlet.
• Hydrogen is mixed with sample and passed through the jet.
• There it is mixed with air and burned in the flame.
• The current flow is recorded as the base line.
• When ionisable materials present in the sample reach the flame and burned,
ions and electrons are formed.
• This raises the current flow. This is sensed as voltage drop, amplified and
recorded.

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• The principle involved in the detector is electron affinity of compounds,
which in turn produce changes in the current which is recorded.
Construction
• It consists of two electrodes.
• The surface of one electrode has radioactive sources which include tritium
or nickel-63 and acts as cathode.
• The other collector electrode is an anode that collects the electrons.

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Electron Capture Detector

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Working
• The radio isotope emits high energy electrons (B-Particles) continuously.
• These electrons bombard the carriers gas and produce thermal electrons,
positive ions and radicals.
• Now voltage is applied as a sequence of narrow pulses to the electron
capture cell that allows the collection of thermal electrons in the collector
electrode.
• This creates the standing current or baseline signal when only carrier gas is
passing through the detector.
• When sample is passed, the compounds react with thermal electrons and
produce negative ions.
• The decrease in thermal electrons by recombination results in decrease in
detector current which is recorded and analysed.

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• The detector is mainly used for determination of volatile sulfur and
phosphorous compounds.
• This uses a flame in which the sample is introduced.
• Air and hydrogen are also supplied with carriers gas.
• The flame is viewed as phosphorous and sulfur forms species that emits band
emissions to give the detector response.

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• The column effluents are burned in a furnace to form species.
• These molecular species are readily ionized and cause electrolytic
conductivity of deionized water which is observed.
• The conductivity of water is regenerated using an ion exchange column
that removes the analyte ions.

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In addition to the above detector some other detectors are also used.
i. Thermionic Emission Detector
• This resembles a FID except that it has a non-volatile bead of rubidium
silicate above the flame tip.
• It uses a low temperatures flame, fuel-poor hydrogen plasma.
• It response to both nitrogen and phosphorous containing compounds with
small hydrogen flow.
ii. Photo Ionisation Detectors
• In this detector UV radiator is mainly used to ionize the sample
components.
• The ions thus produced are collected at an electrode of positive charge.
• The change in current is measured.
• The ionization potential of the compounds should be lower than the lamp
energy to give response.

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• When a compound is not suitable for analysis using GC, it is chemically
modified to produce a new compound or derivative of the parent compound
this process is termed as Derivatization.
This process is often desirable for the following reasons :
1. To improve thermal stability of compounds.
2. The volatility of the compounds can be adjusted so that the separation
properties can be changed and effective resolution can be achieved.
3. To convert the compound suitable for detection.
• The most commonly used derivatization procedures involve the "substitution
of active hydrogens" on the compound to be derivatized with a variety of
functional groups.
• These functional groups impart the desired characteristics to the compound,
while eliminating the adverse effects.
• Generally the compound is derivatized with suitable reagent and the resultant
mixture is injected directly into the Gas Chromatography.
Derivatization

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For Example
1. N, O-bis (trimethyl silyl) acetamide or N, O-bis (trimethyl silyl) trifluro
acetamide are used to convert one active hydrogen in polar groups such as
OH, COOH, NH2, NH, and SH to silylated group. (-0-si(CH3)3).
2. Androsterone exhibits poor peak shape and poor separation by GC. It
contains a hydroxyl group and a carbonyl group. It is derivatised using
trimethylsilylimidazole (TMSI) a strong silyl donor silylation, active
hydrogen on OH, is replaced to produce a trimethylsilyl (TMS) derivative.
The carbonyl group is reacted with another derivatizing reagent
Methoxyamine to give forming an oxime derivative (CH3ON). Oxime
derivatives not only improve chromatographic performance, but also alter
GC separations.

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• This is the method to quantify the analytes from the mixture after separation
using GC.
• Quantitation can be done by different methods and the analysis is termed as
quantitative analysis.
• Quantitative analysis is performed by calculating the area of peaks from
chromatogram.
• Area under the peak of single component is considered to be proportional to
the quantity of the detected component.
The quantitative analysis can be done by following methods :
1. Area normalisation method or area percent method
2. Corrected area normalisation method
3. Internal standard method
4. External standard method
Quantitation

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• The normalization method is the easiest method which requires no
reference standards or calibration solutions to be prepared.
• In this method, the ratio of individual peak area and the sum of all peak
area is taken into consideration.
• The sum of all peak areas is assumed as 100 %.
• Then the percentage of each peak is calculated in terms of 100 %.

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For example
If a chromatogram consists of three peaks A, B and C with areas of 10, 30 and
50 respectively, and the sum of all peaks is 90,
the concentration of A is (10/90) x 100 = 11.11%.
Similarly Concentration of B = (30/90) x 100 = 33.33 %.

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• In area normalisation method it is assumed that the detector response is same
for all peaks.
• If the detector response is not same for all peaks then corrected area
normalisation method is followed.
• Initially relative response factor (RRF) is determined using known amount of
standard.
• This RRF is used to determine the corrected peak area for each peak of
samples which is related to the concentration.

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• In this method a reference standard is added in a constant amount to samples
and calibration standards and analysed.
• This reference standard is termed as internal standard.
• The ratio of analyte peak area to internal standard peak area is used as
analytical parameter.
• An internal standard (IS) is a compound that is similar in physical and
chemical characteristics to the sample being analysed.
• It must be inert to the sample and must not react with the sample or any
solvent used to dilute or prepare it for GC.
• The purpose of the internal standard is to behave similarly to the analyte but
to provide a signal that can be distinguished from that of the analyte, The
peak of the internal standard must not overlap with the peaks of the analytes.

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• In this method, a reference standard is chosen and it is chromatographed
separately from the sample, maintaining the chromatographic conditions
constant. The results of two chromatograms are compared.
• To reduce the effect of any changes in the operating conditions the sample and
reference solutions can be chromatographed alternately.
• The data from the reference chromatograms run before and after the sample
are then used for calculating results of each assay.
• The reference standard (or standards) can be chosen to be the same as the
solute (or solutes) in the sample. This eliminates the need for response factors.
• In addition, the external standard(s) can be made up to have concentration(s)
closely similar to the component(s) of the sample, thus, errors is reduced.
• The sample must fall within a range bracketed by the calibration solution.

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Applications
• Qualitative Analysis
GC is used for qualitative analysis in separation and identification of mixtures by
comparing the retention time of sample compounds with the standard
compounds.
In addition it is used to check the purity of the samples in comparison with the
standards from the appearance of additional peaks which may be of impurities
present in the sample.
• Quantitative Analysis
It is widely applied for the quantitative analysis. The various methods of
quantitative analysis have been discussed under section.
• In Pharmaceutical analysis
Gas Chromatography is regularly used in pharmaceutical industries for drug
analysis. It is conveniently used for drugs with volatile nature.
It is useful for the analysis of antibiotics, antiviral agents, anticancer agents,
Hypnotic and sedatives etc.

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• Determination of Pesticides
GC is a useful tool for the identification and determination of pesticidal residue in
food products, aquaculture products, agricultural products.
• In Food Industry
GC has been employed in food industry for separation and identification of lipids,
carbohydrates, proteins, colorants, flavours and preservatives, vitamins, steroids
and trace elements also can be done using Gas Chromatography.
Sugar contents in the dairy products & determining free cholesterol in milk fat.
• In Forensic sciences
GC is an useful tool for determination of steroid drugs in the blood samples, used
in athletes and sports activities. In forensic sciences, the biological matrices such
as blood plasma, serum and urine samples can be analysed for the drug content.
• In Air monitoring
It is used for the analysis of Toluene, Ethylbenzene, o-xylene and Cumene in air.
Volatile organic compounds are a cause of concern for human health due to their
increased presence in the indoor environment.
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• In volatile mixture separation
GC can be used in many different fields such as pharmaceuticals cosmetics and
even environmental toxins. Since the samples have to be volatile, human breath,
blood, saliva and other secretions containing large amounts of organic volatiles
can be easily analyzed using GC.
• In Petroleum Industry
Gas chromatography has been used in analysis of crude petroleum products,
fractions gasoline, waxes, LPG, Sulphur and nitrogen compounds and reformats.
• In Biochemical and Clinical
The technique is especially useful for applications involving body components of
all types. Blood gases, estrogens, vanillin, mandelic acid etc. have been
determined and analyzed in clinical medicine.
• In Cosmetic and Perfume Fields
Gas chromatography is helpful in determining the composition of various
cosmetics, the quality of ingredients and the components of suitable fragrances.
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