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Unit 4. Chromatography-II
4.1 Gas Chromatography

In gas chromatography, the components of a vaporized sample are separated as a consequence of

being partitioned between a mobile gaseous phase and a liquid or a solid stationary phase held in
a column. In performing a gas chromatographic separation, the sample is vaporized and injected
onto the head of a chromatographic column. Elution is brought about by the flow of an inert
gaseous mobile phase. In contrast to most other types of chromatography, the mobile phase does
not interact with molecules of the analytes; its only function is to transport the analyte through
the column.
There are two types of gas chromatography: gas-liquid chromatography (GLC) and
gas-solid chromatography (GSC)

i. Gas-liquid chromatography (GLC)

Gas-liquid chromatography is based on partitioning of the analytes between a gaseous mobile
phase and a liquid stationary phase immobilized on the surface of an inert solid packing or on
the walls of capillary tubing.

ii. Gas-solid chromatography (GSC)

Gas-solid chromatography is based on a solid stationary phase in which retention of analytes
occurs because of physical adsorption.

4.1.1. History of Gas Chromatography

The concept of gas-liquid chromatography was first enunciated in 1941 by Martin and Synge,
who were also responsible for the development of liquid-liquid partition chromatography. More
than a decade was to elapse, however before the value of gas-liquid chromatography was
demonstrated experimentally and this technique began to be used as routine laboratory tool. In
1955, the first commercial apparatus for gas-liquid chromatography appeared on the market.

4.1.2 Principle of Gas Chromatography

In gas chromatography the separation of mixtures ranging from micrograms to nanograms
quantities takes place under the influence of gas mobile phase. The separation is achieved due to
adsorption rate (in case of GSC) or due to relative solubility (in case of GLC).
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The mobile phase is called the carrier gas. The stationary phase is either inert solid adsorbent or a
non-volatile inert liquid supported on some solid material. The sample is injected through an
injection port, which is heated in an oven in order to vaporize the sample. The carrier gas carries
the sample through the column and detector. In order to keep the sample components in
vaporized form, all these components of the instrument are kept in high temperature. A
schematic for a gas chromatograph is shown in Fig. 4.1.

Figure 4.1 Schematic for simple gas chromatography

4.2 Instrument Parts of Gas Chromatography

The basic components of a typical instrument for performing gas chromatography are shown in
Figure 4.2 and are described briefly.

4.2.1. Carrier Gas System

The mobile-phase gas in gas chromatography is called the carrier gas and must be chemically
inert. Helium is the most common mobile-phase gas, although argon, nitrogen, and hydrogen are
also used. These gases are available in pressurized tanks. Pressure regulators, gauges, and flow
meters are required to control the flow rate of the gas.
Flow rate of carrier gas is normally controlled by a two –stage pressure regulator at the gas
cylinder and some sort of pressure regulator or flow regulator mounted in the chromatograph.
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Inlet pressures usually range from 10 to 50 psi greater than room pressure, which leads to flow
rate of 25 to 150 mL/min with packed columns and 1 to 25 mL/min for open tubular column.

Figure 4.2 Block diagram of a typical gas chromatograph.

4.2.2. Sample Injection System

Column efficiency requires that the sample be of suitable size and be introduced as a “plug” of
vapor. It is important that samples are introduced into the flowing gas stream quickly and in as
small volumes as possible to obtain good separational performance. Slow injection or oversized
sample cause poor resolution. Calibrated microsyringes are used to inject liquid sample through a
rubber or silicone diaphragm, or septum, into a heated sample port located at the head of the
column. The sample port is ordinarily kept at about 50 oC greater than the boiling point of least
volatile components of the sample.
Liquid sample injection ports typically allow a volume of 0.1-20 µL sample to be injected.
Gaseous samples are normally injected using gas syringes and a special gas sampling valve.

4.2.3. Gas Chromatographic Columns

Chromatographic columns vary in length from less than 2 m to 50 m or more. They are
constructed of stainless steel, glass, fused silica, or Teflon. To fit into an oven for thermostating,
they are usually formed into coils having diameters of 10 to 30 cm.
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Column temperature is an important variable that must be controlled to a few tenths of a degree
for precise work. Thus, the column is ordinarily housed in a thermostated oven. The optimum
column temperature depends on the boiling point of the sample and the degree of separation
required. Roughly, a temperature equal to or slightly above the average boiling point of a sample
results in a reasonable elution time (2 to 30 min).
For samples with a broad boiling range, it is often desirable to employ temperature
programming, whereby the column temperature is increased either continuously or in steps as
the separation proceeds.

Types of columns
The columns used in GC may be of following types.
i. Packed Columns
Modern packed columns are fabricated from glass or metal tubing. They are typically 2 to 3 m
long and have inside diameters of 2 to 4 mm. These tubes are densely packed with a uniform,
finely divided packing material, or solid support, that is coated with a thin layer (0.05 to 1 mm)
of the stationary liquid phase. The tubes are usually formed as coils with diameters of roughly 15
cm so that they can be conveniently placed in a temperature-controlled oven.

Criteria for solid adsorbent/solid support

The packing, or solid support, in a packed column serves to hold the liquid stationary phase in
place so that as large a surface area as possible is exposed to the mobile phase.
Following requirements must be fulfilled by the adsorbent used for GSC:

a) It must be thermally stable at the temperature at which the column is being operated.
b) It should be inert during analysis.
c) It should have uniform particle size.

Solid as stationary phase and solid support:

There are different kinds of solid materials which are used as stationary phase or as support for
liquid stationary phase. These include Alumina, Silica, Graphite and Diatomaceous earth.
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Diatomaceous Earth:
These are the skeletal remains of the unicellular algae and known as diatoms. Diatomaceous
earth consists of hydrated Silica group. The large surface area of Diatomaceous earth is mainly
ideal for GLC on solid support.

Criteria for liquid stationary phase:

a) The liquid must be non-volatile at analysis temperature.

b) Polarity should be selected according to sample nature.
c) The liquid should be highly pure.

Polar sample components are retained longer in polar liquid stationary phase as compared with
non-polar component and separation is based on the polarity of the components. While in case of
non-polar stationary liquid phase, the components are separated according to their relative
solubility in the liquid phase.

ii. Capillary or Open Tubular Columns

Capillary columns are also called open tubular columns because of the open flow path through
them. In such capillary columns, the stationary phase was a film of liquid a few tenths of a
micrometer thick that uniformly coated the interior of a capillary tubing.
They are of two basic types: wall-coated open tubular (WCOT) and support-coated open
tubular (SCOT).
a. Wall-coated open tubular (WCOT) Columns
Wall-coated columns are capillary tubes coated with a thin layer of the liquid stationary phase.
Early WCOT columns were constructed of stainless steel, aluminum, copper, or plastic.
Subsequently, glass was used.
b. Support-coated open tubular (SCOT) Columns
In support-coated open tubular columns, the inner surface of the capillary is lined with a thin
film (30 mm) of a solid support material, such as diatomaceous earth, on which the liquid
stationary phase is adsorbed. This type of column holds several times as much stationary phase
as does a wall-coated column and thus has a greater sample capacity. Generally, the efficiency of
a SCOT column is less than that of a WCOT column but significantly greater than that of a
packed column.
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4.2.4. Gas Chromatographic Detectors

Dozens of detectors have been investigated and used with gas chromatographic separations. The
most widely used detectors are described in the paragraphs that follow.
i. Flame Ionization Detectors
The flame ionization detector (FID) is the most widely used and generally applicable detector for
gas chromatography. With a FID, such as that shown in Figure 4.3, effluent from the column is
directed into a small air/hydrogen flame. Most organic compounds produce ions and electrons
when pyrolyzed at the temperature of an air/ hydrogen flame. These compounds are detected by
monitoring the current produced by collecting the ions and electrons. A few hundred volts
applied between the burner tip and a collector electrode located above the flame serves to collect
the ions and electrons. The resulting current (10–12 A) is then measured with a sensitive
picoammeter. The amount of current produced is proportional to the number of ions striking the
collector electrode.

The number of positive ions is related to the number of carbon atoms present in the compound.
FID responds only to those compounds which can easily be ionized in the air-Hydrogen flame.
These properties make the flame ionization detector a most useful general detector for the
analysis of most organic samples including those that are contaminated with water and the oxides
of nitrogen and sulfur. It does not respond to the Inorganic compounds containing N2, CO2, H2O
and O2.

Advantages of FID
a. The FID exhibits a high sensitivity (10–13 g/s).
b. Large linear response range (107).
c. It is generally rugged and easy to use.

Disadvantages
a. It destroys the sample during the combustion step and destructive in nature.
b. Inorganic compounds containing N2, O2, and CO2 cannot be detected.
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Figure 4.3. A typical flame ionization detector

ii. Thermal Conductivity Detectors

Thermal conductivity is the measure of ability of a substance to conduct heat. TCD is a device
that monitors the rate of heat transferred away from an electrically heated wire by the column
effluents. The thermal conductivity detector (TCD), which was one of the earliest detectors for
gas chromatography, still finds wide application.
This device consists of an electrically heated source whose temperature at constant electric
power depends on the thermal conductivity of the surrounding gas. The heated element may be a
fine platinum, gold, or tungsten wire or, alternatively, a small thermistor. The electrical
resistance of this element depends on the thermal conductivity of the gas.
As a gas is passed over a heated filament wire, the temperature and thus the resistance of the
wire will vary according to the thermal conductivity of the gas. The pure carrier gas is passed
over one filament, and the effluents gas containing the sample constituents is passed over
another. These filaments are in opposite arms of a Wheatstone bridge circuit that measures the
difference in their resistance. So long as there is no sample gas in the effluent, the resistance of
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the wire will be the same. But whenever a sample component is eluted with the carrier gas, a
small resistance change will occur in the effluent arm. The change in the resistance, which is
proportional to the concentration of the sample components in the carrier gas, is registered on the
recorder. The TCD diagram is shown in figure 4.4.

Figure 4.4. Schematic of (a) a thermal conductivity detector cell and (b) an arrangement of two
sample detector cells (R2 and R3) and two reference detector cells (R1 and R4).

Choice of carrier gas

Hydrogen and helium carrier gases are preferred with thermal conductivity detectors because
they have a very high thermal conductivity compared with most other gases, and so the largest
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change in the resistance occurs in the presence of sample components gases (helium is preferred
for safety reasons).
Advantages of TCD
a. TCD are simple, having large linear range dynamic range.
b. It response to both organic and inorganic species.
c. It IS nondestructive in nature, which permits collection of solutes after detection.
Disadvantages
a. The chief limitation of this detector is its relatively low sensitivity (108 g/s solute/mL
carrier gas).

iii. Electron Capture Detectors (ECD)

There are certain functional groups that have affinity to take electrons. ECD is based upon taking
up of electrons by these functional groups. In ECD, the carrier gas passes through a β-source
which is usually 63Ni. The energy produced by these β-sources is sufficient to produce positive
carrier gas ions and free electrons. These electrons are attracted towards a positively charged
collector electrode which results a current flow in the external circuit.
Now if a compound containing electron capturing functional group enters the detector along with
carrier gas then some of the electrons are captured by these ions and the result is decrease in
current. This decrease appears as peak on the chromatogram. The ECD utilizes the change in the
electrical conductivity of carrier gas caused by the electrons capturing functional groups.

Usually, N2 or Ar is used as carrier gas which produces high number of thermal electrons.

Advantages:
a. ECD is highly sensitive to electrophilic molecules e.g. organic molecules containing
electronegative groups like nitro groups, phosphorous, O2 and halogen group.
b. An excellent detector for pesticides is used, mostly for trace environmental elements.
c. A selective detector and is used to detect only those compounds which have affinity towards
electrons.
Drawback
a. The major drawback is the use of radioactive source.
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4.5. Applications of Gas Chromatography

Both qualitative and quantitative analysis can be done by G.C.

4.5.1 Qualitative Analysis:

For the qualitative analysis we have two parameters.
i. Retention Volume ii. Retention Time

The retention volume is given as:

V=f t

“t” is the time, “f” is flow rate of carrier gas and “V” is the amount of carrier gas required to
elute a component from the column.
Under the same experimental condition, the qualitative analysis by G.C can be done by
comparing the retention time or retention volume of sample component with the retention
volume or retention time of standard components.
The retention volume or retention time of the sample component is measured and then compared
with retention volume or retention time of standard measured under the same experimental
conditions.
The qualitative analysis can also be done by the standard addition.
Suppose we have two components “x” and “y” which give two peaks A and B as shown in figure

Now we want to identify that which peak is due to “x” and which is due to “y”.
For this purpose, we run the standard components separately and then compare their retention
time. Another method is “spiking” method. Suppose peak A is due to component “x” and we
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want to confirm it. For this purpose, we add component “x” to the mixture and run the
chromatogram and if there is an increase in the peak height, then it confirms the result.

4.5.2 Quantitative Analysis

For quantitative analysis, there are two parameters.

i. Peak Area ii. Peak Height and quantitative analysis are based on the comparison of
sample and reference standards. The peak height is easier to measure but let satisfactory due to
dependent upon experimental conditions.
In first case, the chromatogram is developed for the working standards and area of each is
measured. After measuring the peak area, it is plotted against concentration of working
standards, then a peak for the sample of unknown concentration is taken, its area is measured and
the concentration is determined from calibration plot.

The second method (peak height) is simple as compared with the first one. In this method the
working standards of different concentrations are prepared and chromatogram is developed for
that. After measuring the peak height for each standard, it is plotted against concentration and a
calibration plot is obtained. After this the peak height for the sample of unknown concentration is
measured and the concentration is determined from the plot.
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4.5.3 Others Applications of G.C

The range of applications of G.C is very wide. The ever change products of daily life have
increased the uses of G.C. Some of the applications of G.C are given as:

i. Biochemical Analysis:

Example of biochemical applications are the forensic analysis of blood and wine-alcohol level.
The sample is taken in small amount diluted and then sealed in a container. It is allowed to
equilibrate for 10-20 minutes and then the produced vapors are transported to the column by
carrier gas and by this the volatile components present in the sample are analyzed.
ii. Food Analysis:

The analysis of food materials included analysis of lipids, proteins, carbohydrates, preservatives,
color contents and vitamins.
 G.C is frequently used for derivatization of lipids to fatty acids, methyl ester of protein by
acid hydrolysis etc. Thus, G.C in quality control for food analysis ensures the quantity of
additives at permitted level.
 Dairy products are also analyzed for volatile components to determine %age fatty acid and
milk contents.
 The composition of volatile components presents in natural food stuff which give them a
characteristic odor and taste can also be analyzed.
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iii. Drug Analysis:

G.C is used both for qualitative and quantitative analysis in case of drug samples for example,
the identification of the active components and contaminants in the sample.
Although some drug samples can be analyzed directly after extraction or by dissolution in a
proper solvent but many require special treatment before analysis like derivatization or
methylation.

iv. Environmental Analysis:

Environmental pollution is great problem for human as the technology progressed population
increased and standard of living have improved, so the environmental problems have also
increased.
The combustion of fuels, deposition of wastes and treatment of crops with pesticides and
herbicides all contribute to the problem.
The uses of G.C technique enable us to study these problems even at trace elemental level.
G.C is also important to the analysis of contaminated ground water caused due to strong
hydrogen bonding between phenols, amines and organic acids etc.

4.6 Gas Chromatography-Mass Spectrometry (GC-MS)

Gas chromatography (GC) is the separation technique of choice for smaller volatile and
semi-volatile organic molecules such as hydrocarbons, alcohols and aromatics, as well as
pesticides, steroids, fatty acids and hormones, making this analytical technique common in many
application areas and industry segments, particularly for food safety and environmental testing.
When combined with the detection power of mass spectrometry (MS), GC-MS can be used to
separate complex mixtures, quantify analytes, identify unknown peaks and determine trace levels
of contamination.
A very powerful tool is the combination of gas chromatography with mass spectrometry, a
technique known as gas chromatography-mass spectrometry (GC-MS).

4.6.1 Principles of Mass Spectrometry

Mass spectrometry is a sophisticated instrumental technique that produces, separates, and detects
ions in the gas phase. The basic components of a mass spectrometer are shown in Figure 4.5. A
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sample with a moderately high vapor pressure is introduced in an inlet system, operated under
vacuum (10-4 to 10-7 torr) and at high temperature up to 300 oC). The sample vaporizes and is
carried to the ionization sources. Nonvolatile compounds may be vaporized by means of a spark
or other source. Analyte molecules are typically neutral and must be ionized. This is
accomplished by various means but typically done by bombarding the sample with high-energy
electrons in an electron-impact source.
The ions are separated in the spectrometer by being accelerated through a mass separator.
Separation of ions occurs on the basis of their mass-to-charge (m/e) ratio.

Figure 4.5 Block diagram of mass spectrometer

4.6.2 Instrumentation of GC-MS

Chromatographic Inlet Systems

Mass spectrometers are often used as detectors in gas-liquid chromatography to quantify analytes
as they elute from the column. Mass spectrometry is an extremely powerful technique in this
context since analytes may be both identified (from their characteristic molecular/ionic masses)
as well as being quantified.

Ionization Sources
There are numerous means of ionizing molecules or elements in a sample, the most appropriate
depending on the nature of the materials and the analytical requirements. The most important
ionization techniques are summarized below.

Electron Impact Ionization (EI)

The most commonly used ionization source is the electron-impact (EI) source. The gaseous
molecules are bombarded by a high-energy beam of electrons, usually 70 eV, generated from a
tungsten filament. An electron that collides with a neutral vaporized analyte molecule may
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impart sufficient energy to remove an electron from the molecule, resulting in a singly charged
ion:

M + e- → M+ + 2e-

Where M is the analyte molecule and M+ is the molecular ion or parent ion. These then
decompose into smaller fragments by further electron bombardment. Figure 4.6 shows a simple
EI spectrum of small molecule, methanol. Usually, peaks are normalized to the one with the
greatest abundance (relative abundance 100%); the largest peak is called the base peak. The
molecular peak is at m/z = 32, the formula weight of CH3OH. The bas peak is at m/z = 31 if from
the CH2OH+ fragment.

Figure 4.6 Electron ionization mass spectrum of methanol.

Chemical Ionization Source (CI)

The EI source is called a “hard source” and may produce too much fragmentation to allow
positive identification of the analyte, and no molecular ion may be present. Chemical ionization
(CI) is a “softer” technique that does not cause much fragmentation and the molecular ion is the
dominant one in CI mass spectra.
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In CI, a reagent gas such as methane, isobutene, or ammonia is introduces into the EI ionization
chamber at a high pressure (large excess, 1 to 10 torr) to react with analyte molecules to form
ions by either a proton or hydride transfer. The chemical ionization process begins by ionization
of the reagent gas. With methane, electron collisions produce CH4+ and CH3+, which further react
with CH4 to form CH5+ and C2H5+.

These react with the sample by transferring a proton (H+) or by extracting a hydride (H-) or
electron, which imparts a +1 charge on the sample molecule:

MH2+ and M+ may fragment to give the mass spectrum. No M+ ion may be observed, but the
molecular weight is readily obtained from the M + H or M-H ions formed.

Mass Analysers
Mass analysers disperse ionized samples according to differences in their mass-to-charge ratio as
they emerge from the ionization source or chamber. Ions are drawn towards and focused through
appositively charged polarized electrode slits to impart kinetic energy and, in this way; ions are
accelerated into the mass analyser. Every effort is taken in the design of the ionization chamber
to ensure that the same kinetic energy is imparted to all of the ions. The ions possess different
masses, however, and will therefore enter the mass analyser with a range of velocities. Ions with
the smallest masses will travel with the greatest velocities and ions with the greatest masses will
travel with the slowest velocities.

4.6.3 Applications of GC-MS

a. Pharmaceutical Applications
 Used for the elucidation of the structure of organic and biological molecules.
 Impurity profiling of pharmaceuticals.
 Identification of compounds in the thin layer and paper chromatograms.
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 Identification of drugs of abuse and metabolites of drugs of abuse in blood, urine and
saliva.
 Testing for the presence of the drugs in blood in race horses and in Olympic athletic.
 Analyser of aerosol particles.
 Determination of pesticides residue in blood.
b. Environmental Monitoring and Clean up
GC-MS is becoming the tool of choice for tracking organic pollutants in the environment.
c. Criminal Forensics
 GC-MS can analyze the particles from a human body in order to help link a criminal to a
crime.
 GC-MS especially useful here as samples often contain very complex matrices and results
used in court.
d. Sports Antidoping Analysis
GC-MS is main tool used in sport anti-doping laboratories to test athletes urine samples for
prohibited performance of enhancing drugs.
e. Food, Beverage and Perfume Analysis
 Food and beverage contain numerous aromatic compounds, some naturally present in the
raw materials and some forming during process.
 GC-MS is extensively used for the analysis of these compounds which include ester, fatty
acid, alcohols, aldehydes, terpenes etc.

Mass Spectrum
The mass spectrum is the plot of mass to charge ration (m/z) of positively charged ions against
their relative abundance. The m/z ratio is taken along the abscissa, while relative abundance is
taken on ordinate.
Base peak
The most intense peak in the mass spectrum is called the base peak. Base peak is the highest
peak it is assigned a relative intensity of 100%.
Molecular Ion peak
The ion formed from a molecule by removal of one electron of lowest ionization potential is
known as molecular ion.
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The molecular ion is detected as mass to charge ratio that corresponds to molecular weight of
molecule. The molecular ion peak gives the molecular weight of compounds. The molecular ion
peak is highest mass number except isotope peak.

Best of Luck
Dr. Muhammad Naeem Khan
Assistant Professor
Department of Chemistry
Allama Iqbal Open University
Islamabad