1. GAS CHROMATOGRAHY (G.

C)

Gas chromatography is a separation technique used for separating volatile

compounds. Volatile compounds are compounds that can easily vaporize at
room temperature. There are basically two types of gas chromatography which
are as follows:

1) Gas liquid chromatography (GLC)

2) Gas solid chromatography (GSC)

In GLC gas is the mobile phase and liquid is the stationary phase, whereas in
GSC uses gas as the mobile phase and solid as a stationary phase. Since gas
chromatography is a gas technique, your sample must be able to be volatile or
converted to vapour phase and sample must also be thermally stable.

Schematic diagram for gas chromatography

MAJOR COMPONENTS OF GAS CHROMATOGRAPHY

1) COLUMN: the column used in gas chromatography is very long and

arranged in a coil, the column used in gas chromatography are of two
types. Packed column and capillary column. Pack column can be made of
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 glass or stainless usually having a length between (1-3 metres) with an
internal diameter ranging from (2-4 mm) whereas the capillary column is
made up of fused quartz, a length between (10-100 mm) and an internal
diameter between (0.1-1 mm) the column is placed in chamber 80 that the
temperature can be maintained.

2) STATIONARY PHASE: The stationary phase is packed in the inner wall of the
column. It is made up of silicon grease or wax which can withstand a high
temperature.

3) MOBILE PHASE: The mobile phase used is usually an inner gas like helium
or un-reactive gas such as nitrogen kept in a cylinder which is connected
with the column by a molecular sieve. The molecular sieve separates or
removes unwanted hydrocarbon water vapour and oxygen that may
interfere with a sample during analysis.
4) DETECTOR: At the end of a column there is a detector connected. This
detector detects the sample which sends information to the computer
connected to it and hence produces result in form of a chromograph.

WORKING PPRINCIPLES OF GAS CHROMATOGRAPHY

Sample which is to be separated is mixed with an appropriate volatile solvent

sample such as Heptane, Aceton or Methanol. Just before the column there is
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a spectrum that allows infection of the sample. The temperature of the
infection region is kept 20 to 50oC higher than column as this allows rapid
volatilization of the sample. Once the sample is volatized, it passes down the
column where the separation occurs.

During analysis, the temperature of the column is kept between 150-300 oC and
separation occurs based on the interaction of molecules between the mobile
phase and the stationary phase. The less volatile molecules interact more with
stationary phase and move slowly while more volatile molecules interacts
more with the mobile phase and moves faster down the column. Once the
separation is completed, the detection is done with a detector at the end of
the column. The common detect used in gas chromatography is F10 (Flame
ionization) detector. It has three inlets, one for the carrier gas which comes
from the column, one for hydrogen and the other for oxygen.

Above the flame ionization detector there is an ignitor that ignites hydrogen
and oxygen to produce a flame, when sample molecules reaches the frame,
they get ionized and electrons are released. Across the flame they are two
electrodes with a positive and negative charge, the electrodes detests the
electrons produced or generated by the ionization of the sample, electrons are
detected in the form of current, which amplified and detected by the
computer. When the sample is detected, the computer gives peak with respect
to the retention time of the sample, the area under the peak gives information
about the concentration of the sample. If the concentration is high, the area
under the peak will be high and if the concentration is less, then the area will
be less. For the detection of unknown samples we need to have standards.

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 Schematic diagram of an FID

USES AND APPLICATION OF GC

1) Alkaloid Chemistry: GC is a similar method to HPLC. It provides quick and

easy way of determining alkaloids in a mixture. The only requirement is
some degree of stability at the temperature necessary to maintain the
substance in the gas state.
2) Drinking water detection: (GC) is a common type of chromatography used
in analysing compounds that can be vaporized without decomposition.
Typical uses include testing the purity of a particular substance; also help in
identifying compounds of mixture.
3) Moisture Loss, gain migration in foods and its impact on food quality. GC
has been applied to the determination of water content-dried products.
However, water has been extracted by organic solvents before analysis and
the sample has to be homogenous.
4) Antineoplastic Drugs: GC, combined with nitrogen phosphorus detector or
mass spectrometry can be used in of specific neoplastic drugs.
5) Controlled Atmosphere Storage: GC is used to separate and measure
various types of gases, being a very sensitive technique, can analyse small
samples and can be automated, but also requires technical knowledge.
6) Applied in studying Aromatic compounds.

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GAS CHROMATOGRAPHY- MASS SPECTROSCOPE (GC-MS)

Gas chromatography-mass spectroscope is one of the so called hyphenated

analytical techniques. As the name implies, it is actually two techniques that
are combined together to form a single method of analysing mixtures of
chemical.
Gas chromatography separate the component of a mixture and mass
spectroscopy, characterises each of the components individually. By combining
the two techniques, an analytical chemist can both qualitatively and
quantitatively evaluate samples containing a number of chemicals. The uses of
GC-MS are numerous, they are used extensively in the medical,
pharmacological, environmental and law enforcement agencies.
GAS CHROMATOGRAPHY-INFRARED (GC-IR)

The combination of gas chromatography and infrared spectroscopy is a

powerful tool for the characterization of compounds in complex mixtures. GC-
MS is a similar technique but GC-MS is a destructive technique that tears apart
the sample molecules during ionization process then the fragments are used to
characterize.

In GC-IR the molecules are not destroyed but the IR light produced by
molecular vibrations are used to characterize the molecules. IR spectrum yields
information about the whole molecule which allows the characterization of
specific Isomers and functional groups.

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2. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

HPLC is an acronym for high performance or pressure liquid chromatography.

The word chromate means colour and graphy means writing. HPLC was
pioneered by “Christian Friedrich Schonbein (1799-1868). He began by moving
substances through filter paper and Mikhail Semyonorich Tsvet (1872-(1919)
who separated plant pigment in calcium carbonate column.

HPLC is the separation of compounds on a stationary phase column, HPLC can

also be referred to as an advancement of the classical column chromatography
which has two problems

1. Column chromatography relies on gravity and gravity takes a lot of time to

take the sample from the stationary phase to the mobile phase
2. They are relatively coarse

HPLC as an advancement has solve the above problems hence

1. It uses pressure that is very high to take sample from the stationary phase
to the mobile phase.
2. HPLC movement is more versatile than colour and gas chromatography.

Unlike gas chromatography whose mobile phase is gas, HPLC the liquid that
constitute the mobile phase could be a single binary, ternary, quaternary
solvent. The stationary could be depending on the following:

1. Unmodified solid absorbent

2. Chemically modified absorbent
3. Ion-exchange
4. Size exclusion materials

Types of HPLC

There are two most common types of HPLC, they are as follows

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NORMAL-PHASE HPLC: The column is filled with tiny silica particle and non-
polar solvent, for example, hexane. A typical column has an internal diameter
of 6mm or smaller and a length of 250-250mm. Non-polar compounds in the
mixture will pass more quickly through the column, as polar compounds will
stick longer to the polar silica than non-polar compound will.

REVERSED-PHASE HPLC: the column size is the same. The column is filled with
silica particles which are modified to make them non-polar. This is done by
attaching long hydrocarbon (8-18 carbon atoms) to the surface. A polar solvent
is used for example, a mixture of water and alcohol such as methanol. Polar
compounds in the mixture will pass more quickly through the column because
a strong attraction occurs between the polar solvent and the polar molecules
in the mixture.

Non-polar molecules are slowed down on their way through the column. They
form varying degrees of attraction with the hydrocarbon groups principally
through vander waal dispersion forces and hydrophobic interactions. They are
also less soluble in the aqueous mobile phase component facilitating their
interactions with the hydrocarbon groups. The Reverse-phase HPLC is the most
common used form of HPLC.S

Schematic diagram of HPLC

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COMPONENTS OF A HPLC SYSTEM

1. THE MOBILE PHASE: This is the solvent that runs continuously through the
system and pushes the sample through the column, the solvent is
contained in a reservoir located at a higher elevation than the pump in
other to maintain a slight positive head pressure on the pump inlet. A
solvent filter removes any particles that could potentially damage the
system sensitive components.
2. AN AUTO SAMPLER: An auto sampler stores the sample vials or a plate in a
temperature controlled environment and infect the desired sample when
instructed by the data system, when a separation need to be performed.
The auto sampler switches the valve in order to fill the sample loop with
the sample for analysis, and then switches back to infect the sample, An
auto sampler allows the user to perform multiple sequences of runs
unattended.
3. A COLUMN: The column is packed with a stationary phase that separates
the sample, a non-polar silica base phase is the most common phase for
reverse-phase HPLC.
4. ONE /MORE DETECTOR: the detector receives the result of the sample
separation from the column and monitors the physical property which
changes as the sample elutes.
5. A CHROMATOGRAPHY DATA SYSTEM (CDA): The data system translates
the signal from the detector into a chromatographic spectrum that
provides qualitative an quantitative data about the sample. The data
system allows complete control of the pump auto sampler and detector, all
of the instrument parameters automated run sequences and data
collection can be controlled by the data system.

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UHPLC

UHPLC- This means ultra high performance liquid chromatography is a

technique that

 Increase separation efficiency

 Improve resolution
 Provides shorter analysis time
 Provides lower operating cost

It does this by using HPLC column with mean particle size diameter less than
two microns along with the new commercially available instrumentation
capable of driving the illuminated through the columns at subsequent higher
pressure, this allows optional linear vehicles to be reached for smaller particles
size column.

HPLC-MS

High performance liquid chromatography-mass spectrometry is an extremely

versatile instrumental technique whose root lies in the application of more
traditional liquid chromatography to theories and instrumentation that were
originally developed for gas chromatography (GC). As the name suggest the
instrument comprises a high performance liquid chromatography (HPLC)
attached via a suitable interface to a mass spectrometer (MS). The primary
advantage HPLC/MS has over GC/MS is that it is capable of analysing a much
wider range of components. Compounds that are thermally labile, exhibit high
polarity or have a high molecular mass may all be analysed using HPLC/MS,
even proteins may be routinely analysed. Solutions derived from sample of
interest are onto an HPLC column that comprises a narrow stainless steel tube
(usually 150mm length and 2mm internal diameter or smaller) packed with
fine chemically modified silica particles. Compounds are separated on the basis
of their relative interaction with the chemical coating of these particles
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(Stationary Phase) and the solvent eluting through the column (Mobile Phase).
Components eluting from the chromatographic column are then introduced to
the mass spectrometer via a specialized interface. The two most common
interfaces used to HPLC/MS are the electrospray ionization and the
atmospheric pressure chemical ionization interfaces

Schematic diagram of HPLC-MS

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