Chromatography
Chromatography is a non-destructive process for resolving a complex
mixture into its individual components. It’s a separation procedure and
the separated entities are identified by other analytical techniques like
UV-visible, IR (Infrared), NMR (nuclear magnetic resonance), Mass
spectrometry etc.
Chromatography was invented by Russian botanist Mikhail Tswett. The
term chromatography came from Greek word chroma (means color)
and graphein (means write).
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Definition:
Chromatography is a technique in which the components of a mixture are
separated based upon the rates at which they are carried through the stationery
phase by a gaseous or liquid mobile phase.
or
Chromatography is defined as the process of separation of the individual
components of a mixture based on their relative affinities towards stationary
and mobile phases.
According to IUPAC, chromatography is a physical method of separation in
which the components to be separated are distributed between two phases,
one of which is stationary, the other (the mobile phase) moves in a definite
direction.
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Principle of Chromatographic separation
The samples are subjected to flow by mobile phase onto or through the
stable stationary phase. The separation of components of the mixture
takes place based on their relative affinity towards the two phases
during their travel. The fraction with greater affinity to stationary phase
travels slower and shorter, while that with less affinity travels faster
and longer.
In partition chromatography, on moving with mobile phase the
components of the sample equilibrate between the mobile phase and
the stationary phase.
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Classification of Chromatography Methods
1. Planar Chromatography
2. Columnar Chromatography
Planar Chromatography:
In this chromatography the stationary phase is supported on a flat place
or in the pores of a paper. Here the mobile phase moves through the
stationary phase by capillary action or under intense of gravity.
This chromatographic method includes-
i. Paper chromatography
ii. Thin layer chromatography (TLC)
iii. High performance thin layer chromatography (HPTLC)
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Columnar Chromatography
In this method, the stationary phase is held in a narrow tube and the mobile
phase is forced through the tube under pressure or by gravity. Columnar
Chromatography methods fall in to following categories based upon the
nature of mobile phase.
A. Liquid Chromatography (LC) (where mobile phase is a liquid)
These subdivisions are based on the nature of the stationary phase and by the
types of equilibria between phases. e.g.
i. Column chromatography
(a) Liquid –Liquid or Partition chromatography
(b) Liquid – Solid or adsorption chromatography
ii. High performance liquid chromatography (HPLC)
iii. Ion– exchange chromatography
B. Gas Chromatography (GC) (where mobile phase is a gas)
i. Gas – Liquid chromatography (GLC)
iii. Gas - Solid chromatography
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Some important terms
Stationery phase: The stationery phase in Chromatography is a phase that is fixed in place
either on a planar surface or in a column . The stationary phase may be solid or liquid.
Mobile phase: The mobile phase in Chromatography is a phase that moves over or through
the stationary phase, carrying the analytes with it. The mobile phase may be gas or liquid.
Elution: Elution is a process in which components are washed through a stationary phase by
the movement of a mobile phase.
Eluent: An eluent is a solvent used to carry the components of a mixture through the
stationary phase.
Chromatogram: Chromatogram is the visual output
of a chromatographic separation.
It can be defined as a graph showing the quantity of a
substance leaving a chromatography column as a
function of time.
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Paper Chromatography
Paper Chromatography is one of the types of chromatography procedures which runs on a
piece of specialized paper.
Principle:
The principle involved is partition chromatography, where the substances are distributed
or partitioned between to liquid phases.
One phase is the water which is held in pores of filter paper used and other phase is that
of mobile phase which moves over the paper.
The compounds in the mixture get separated due to differences in their affinity towards
water (in stationary phase) and mobile phase (solvents) during the movement of mobile
phase under the capillary action of pores in the paper.
The principle can also be adsorption chromatography between solid and liquid phases,
where in the stationary phase is the solid surface of paper and the liquid phase is of
mobile phase.
But most of the applications of paper chromatography work on the principle of partition
chromatography.
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Uses/applications of paper chromatography
Paper chromatography is specially used for separation of
mixture having polar and non polar compounds.
For separation of amino acids.
To determine organic compounds, biochemicals in urine etc.
In pharma sector, for determination of hormones, drugs.
Sometimes used for evaluation of inorganic compounds like
salts and complexes.
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Thin Layer Chromatography (TLC):
Principle:
¤ This technique involves the use of an inert piece of glass, plastic or metal.
¤ The glass plate is coated with a thin layer of silica gel that acts as the
stationary phase.
¤ A drop of solution to be analyzed is added to the TLC glass plate.
¤ The sheet is then placed in a glass chamber containing a solvent
(mobile phase).
¤ Due to capillary action, the solvent moves upwards and carries the
molecules of the solution to be detected at different rates.
¤ The components of the solution will appear in the form of a series of
spots at various locations on the plate.
¤ Calculations are made based on ratio of the distance that the
substance travels to the distance that the solvent travels up the plate.
Components of TLC System:
TLC System consists of
TLC plates preferably ready made with stationary phase. These are stable and
chemically inert plates on to whose surface a thin layer of stationary phase is
applied. The stationary phase on the plates is of uniform thickness and
consists of fine particle size.
TLC chamber: This is used for the development of TLC plate. The chamber
maintains uniform environment inside for proper development of spots. It also
prevents the evaporation of solvents and keep the process dust free.
Mobile phase: This comprises of a solvent or solvent mixture. The mobile
phase used should be particulate free and of highest purity for proper
development of TLC spots. The solvents recommended are chemically inert
with the sample and stationary phase.
A filter paper moistened in the mobile phase, is placed inside the chamber.
This helps uniform rise in mobile phase over the length stationary phase
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Advantages of TLC
It is simple process with short development time.
It helps in visualization of separated compound spots easily.
The method helps to identify the individual compounds.
It helps in isolation of most of the compounds.
The separation process is faster and the selectivity for compounds is higher.
The purity standards of the given sample can be assessed easily.
It is a cheaper chromatographic technique.
Applications
¤ To check purity of given samples.
¤ Identification of compounds like acids, alcohols, proteins, alkaloids, amines,
antibiotics etc.
¤ To evaluate reaction process by assessment of intermediates, reaction course etc.
¤ To purify samples i.e., for purification process.
¤ To keep a check on the performance of other separation processes.
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Column Chromatography
Column chromatography is basically a type of adsorption chromatography.
Here the separation of components depends upon the extent of adsorption to
stationary phase.
Here the stationary phase is a solid material packed in a vertical column made of
glass or metal.
Principle:
In this technique a powdered adsorbent, (silica (SiO2) or alumina (Al2O3) is placed
in a vertical glass column as the stationary phase,.
The mixture to be analyzed is placed on top of this vertical glass column.
The solvent forming the mobile phase is poured from the top of the column and
flows down.
As the solvent flows, the solvent elutes the sample through the column and the
components of the mixture begin to flow at their respective rates down the
column.
The solute molecules adsorbProf.toDr.the
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column in a reversible manner. 14
Procedure:
The stationary phase material is suitably moistened with
mobile phase and packed sufficiently in the column with a
cotton or asbestos pad at the bottom.
The sample is placed on the top of packed stationary phase
with a second cotton or asbestos pad in between.
The mobile phase flows through the column taking the sample
with.
The separation of sample components depending on their
interaction between the stationary phase and the mobile phase
takes place.
Individual components along with mobile phase (elute) are
collected in a beaker placed at the bottom of column.
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Applications
Column chromatography is best suited to separate active
substances from plant materials.
In separation of compounds after organic synthesis to
obtain desired molecule.
To separate or purify natural compound mixtures like
alkaloids, glycosides.
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Gas Chromatography (GC)
Principle:
This technique involves the packing of a stationary phase in a long,
narrow glass/metallic column.
The stationary phase is a high-boiling liquid into which the mixture to
be analyzed is loaded with the help of a syringe.
An inert carrier gas (Helium or Nitrogen) acts as the mobile phase,
which flows through the column with the stationary phase.
Once the gas is passed, the components of the mixture is begun to
distribute between the stationary high-boiling liquid and the inert
gas.
Then the components travel themselves through the detector and
reveal themselves in the form of different peaks on a recorder.
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Instrumentation:
The basic instrumentation for GC includes:
ʘ A carrier gas cylinder with regulator
ʘ A flow controller for the gas
ʘ An injection port for introducing the sample
ʘ A column
ʘ A detector
ʘ A recorder
In GC, the injection port, column and detector are to be heated to a pre-
specified temperature. Since the mobile phase here is a gas (carrier gas) the
components present in the analyte mixture should be vaporized, so that it can
be effectively carried through
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Fig. A typical schematic diagram of Gas Chromatography
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The injection port, column oven and detector are hot zones.
The success of this technique requires the appropriate selection
of the column and the temperature conditions at which the
column to be maintained throughout the analysis.
Classification of GC Column:
1. Analytical columns
Packed column
Open-tubular or capillary column
2. Preparative columns.
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Detectors Used in GC:
Detectors play unique role in GC . There are a number of detectors, which vary in
design, sensitivity and selectivity. Few examples and applications are:
1. Thermal Conductivity Detector (TCD)
It works on the basis of the conductivity eluting compound and the carrier gas
It can detects most of the analytes
It is non-destructive
Used with preparative GC
Less sensitive than other detectors
2. Flame Ionization Detector (FID)
Eluting components are ionized (positive and negative) using a hydrogen flame
Positively charged ions are drawn to a collector
Negatively charged ions are attracted to positively charged burner head, this
creates an electric circuit and the signal is amplified
It is restricted for preparative GC
It is used for inorganic substances which do not burn
3. Electron Capture Detector (ECD)
It utilizes the beta emissions of a radioactive source (e.g. nickel-63) to cause
the ionization of the carrier gas molecules
Generated electrons constitute an electrical current
It is useful for the analysis of halogenated pesticide residues found in
environmental and bio-medical samples
It is extremely sensitive
It does not destroy the sample
Used for the preparative work
4. Nitrogen/Phosphorus Detector (NPD)
The design of the detector is same to that of the FID detector
A bead of alkali metal salt is positioned just above the flame
It is also known as ‘Thermionic Detector’
It is useful for the phosphorus and nitrogen containing pesticides
5. Flame Photometric Detector (FPD)
Here a flame photometer is incorporated into the instrument
It is specific for organic compounds containing sulphur or phosphorus
It is very selective and very sensitive
6. Electrolytic Conductivity Detector (ECD)
It is also known as ‘Hall detector’.
It converts the eluting gaseous components into ions in liquid solution
Then measures the electrolytic conductivity of the solution in a conductivity cell.
This detector is used in the analysis of organic halides
It has excellent sensitivity and selectivity
But it is a destructive detector
Note: In recent developments, GC is coupled with other analytical techniques, like Infra-
Red Spectrometry (GC-IR) and Mass Spectrometry (GC-MS). It saves time and reduces the
steps involved for a component to be separated and analyzed.
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GC fitted with Flame Ionization Detector (FID)
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GC fitted with Flame Ionization Detector (FID)
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