CHROMATOGRAPHY.

➢ Chromatography (from Greek chroma "color and graphein "to write")

is the collective term for a set of laboratory techniques for the separation of
mixtures.
➢ The mixture is dissolved in a fluid called the mobile phase (LIQUIDE OR
GAS), which carries it through a structure holding another material called
the stationary phase (SOLID OR LIQUIDE)
➢ The stationary phase is usually in a column, but may take other forms, such
as a planar phase (flat sheet).
➢ The various constituents of the mixture travel at different speeds, causing
them to separate.
➢ The separation is based on differential partitioning between the mobile and
stationary phases.
BACKGROUND KNOWLEDGE:
In 1901, the Russian botanist, Mikhail Tswett, invented adsorption
chromatography during his research on plant pigments. He separated
different colored chlorophyll and carotenoid pigments of leaves by passing
an extract of the leaves through a column of calcium carbonate, alumina, and
sucrose, eluting them with petroleum ether/ethanol mixtures.
DEFINITION:
Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases, one of
which is stationary (stationary phase), while the other (the mobile phase)
moves in a definite direction.
PRINCIPLE OF CHROMATOGRAPHY:
Let us first draw a pictorial representation of a column chromatographic
separation set up.

As depicted above, the analyte is loaded over the silica bed (packed in the
column) and allowed to adhere to the silica. Here, silica acts as the stationary
phase. Solvent (mobile phase) is then made to flow through the silica bed
(under gravity or pressure). The different components of the analyte exhibit
varying degrees of adhesion to the silica (see later), and as a result they
travel at different speeds through the stationary phase as the solvent flows
through it, indicated by the separation of the different bands. The
components that adhere more strongly to the stationary phase travel more
slowly compared to those with a weaker adhesion. Analytical
chromatography can be used to purify compounds ranging from milligram to
gram scale.
Differential affinities (strength of adhesion) of the various components
of the analyte towards the stationary and mobile phase results in the
differential separation of the components. Affinity, in turn, is dictated
by two properties of the molecule: ‘Adsorption’ and ‘Solubility’.

We can define adsorption as the property of how well a component of

the mixture sticks to the stationary phase, while solubility is the
property of how well a component of the mixture dissolves in the
mobile phase.

● Higher the adsorption to the stationary phase, the slower the

molecule will move through the column.
● Higher the solubility in the mobile phase, the faster the molecule
will move through the column.

CLASSIFICATION

Chromatographic methods can be classified in three different ways :-

a) Based on shape of chromatographic beds .e.g.- Planar and column
Chromatography
b) Based on the physical state of the mobile and stationary phase. e.g-
Gas and liquid chromatography.
c) Based on mechanism of separation. e.g.-Ion-exchange
chromatography, partition , affinity and adsorption chromatography.
Classification of Chromatography based on the Chromatographic Bed
Shape
Column Chromatography:
In this type of chromatography, the stationary phase of the setup is placed inside a
tube. Then, the particles of the stationary phase (which is in the solid state) are
made to fill the inside with the tube. An unrestricted, open path is then prepared for
the mobile phase (somewhere along the middle of the tube).
Planar Chromatography:
In this type of chromatography, the stationary phase of the apparatus usually has a
planar shape. Different subcategories of planar chromatography include paper
chromatography (where the stationary phase is a special type of paper) and thin
layer chromatography (usually abbreviated as TLC).

Classification of Chromatography based on the Physical State of the

Mobile Phase
Gas Chromatography: In this type of chromatography, the mobile phase is a
substance that exists in the gaseous state. It can be noted that gas chromatography
is also known as gas-liquid chromatography, and is often abbreviated to GLC. This
type of chromatography almost always involves the use of a packed column.

Liquid Chromatography:
This type of chromatography involves the use of a mobile phase that exists in the
liquid state. Liquid chromatography, often abbreviated to LC, can be carried out
either on a plane or in a column. It can be noted that there exist many subcategories
under liquid chromatography such as high-performance liquid chromatography and
reversed phase liquid chromatography.
Classification of Chromatography based on the Mechanism of the
Separation:
Ion Exchange Chromatography:
This type of chromatography is also known as ion chromatography. Ion exchange
chromatography involves the separation of the components of the mixture via an
ion exchange mechanism. Differently charged components of the mixture are
separated with the help of different ions in this separation technique.
Size Exclusion Chromatography:
This type of chromatography involves the separation of different components of
the mixture based on their sizes. In size exclusion chromatography, components of
the mixture are filtered based on their hydrodynamic volume or hydrodynamic
diameters. It can be noted that size exclusion chromatography is also known as gel
permeation chromatography or gel filtration chromatography.
Partition Chromatography:
It is a technique mainly used for the separation of the components present in the
mixture into two liquid phases that are the original solvent and the solvent coating
utilized in the column.
Types of Partition Chromatography

A- Liquid -Liquid Chromatography – In this type of partition chromatography,

mobile and stationary both phases are taken in liquid state. Although the liquid
stationary phase is coated or supported by solid support.
It has following sub-types –

1) Paper Chromatography – In this type of liquid-liquid chromatography,

liquid stationary phase is taken on chromatography paper made up of
cellulose which is polar substance. Water molecules get trapped on it.

2) Column Chromatography – In this type of liquid-liquid chromatography,

liquid stationary phase is taken on silica gel or alumina cellulose powder.

3) Thin layer Chromatography – In this type of Liquid -Liquid

chromatography, a very thin layer of stationary phase is taken that’s why it is
 known as thin layer chromatography. It is used to separate non-volatile
mixtures.

B- Gas – Liquid Chromatography – In this type of partition chromatography,

mobile phase is an unreactive gas and stationary phase is non-volatile liquid held
on inert solid support. It is also known as vapor-phase chromatography or
Gas-liquid partition chromatography (GLPC).

Adsorption Chromatography:
The stationary phase is a solid on which the sample components are adsorbed. The
mobile phase may be a liquid (liquid–solid chromatography) or a gas (gas–solid
chromatography); the components distribute between the two phases through a
combination of sorption and desorption processes. Thin-layer chromatography
(TLC) is a special example of adsorption chromatography in which the stationary
phase is planar, in the form of a solid supported on an inert plate, and the mobile
phase is a liquid.

In the normal mode of operations of liquid–liquid partition chromatography,

a polar stationary phase (e.g., cyano groups bonded on silica gel) is used, with a
nonpolar mobile phase (e.g., hexane).

When analytes (dissolved in the mobile phase) are introduced into the system,
retention increases with increasing polarity. This is called normal-phase
chromatography.

Principlesof ChromatographicSeparations
While the mechanisms of retention for various types of chromatography differ,
they are all based on the dynamic distribution of an analyte between a fixed
stationary phase and a flowing mobile phase. Each analyte will have a certain
affinity for each phase.
Figure illustrates the separation of components in a mixture on a chromatographic
column. A small volume of sample is placed at the top of the column, which is
filled with particles constituting the stationary phase and the solvent. Rather than
an equilibrium-based “plate view” of chromatography, many hold that a “rate
view” of chromatography to be more rigorous: in this view, the partition ratio is
simply the ratio of the time a solute spends in the stationary phase to that it spends
in the mobile phase.
Chromatographic retention of a compound is often associated with the
establishment of an equilibrium between the stationary phase and the mobile phase,
but in reality, chromatography is a dynamic process, and true equilibrium may not
be reached.
More solvent, functioning as mobile phase, is added to the top of the column and
percolates through the column. The individual components interact with the
stationary phase to different degrees.
The distribution of the analyte between the two phases is governed by many
factors: the temperature, the type of compound, and the stationary and mobile
phases.
TERMINOLOGIES RELATED TO CHROMATOGRAPHY:
1) Chromatogram:
A graphical presentation of detector response, concentration of analyte in the
effluent, or other quantity used as a measure of effluent concentration.

2) Elution:
Elution is the process used to wash components of a mixture through a
chromatography column.
3) Eluent:
The eluent is the mobile phase or the solvent that is passed through the
column.
4) Eluate:
The eluate is the analyte material that emerges from the chromatograph. It
specifically includes both the analytes and solutes passing through the
column, while the eluent is only the carrier.
5) Retention Time:
The retention time (tR) for an analyte is the time between its injection onto a
column and the appearance of its peak as it elutes from the column.
6) Retention Factor:
 The retention factor (k) is the ratio of the amount of analyte in the stationary
phase to the amount in the mobile phase.

where Cs and Cm are the stationary and the mobile phase concentrations.
Above Equation implies,
solutes with a large K value will be retained more strongly
by the stationary phase than those with a small K value.
The result is that the latter will move along the column (be eluted) more
rapidly.
Retention factor in terms of chromatography can also be written as:
The ratio of the time the solute spends in the stationary phase to the time it
spends in the mobile phase:

7) Retention Volume:
Retention volume (Vr) is the volume of mobile phase that is required to elute
a substance from a column.

8) Theoretical plates:
The separation efficiency of a column can be expressed in terms of the
number of theoretical plates in the column.A theoretical plate represents a
single equilibrium step. The more theoretical plates, the greater the resolving
power (the greater the number of equilibrium steps). The more the number
of plates, the more efficient the column. The number of plates, N, for a
solute eluting from a column is given by:
9) Plate Height:
The plate height, H, is the length of a column, L, divided by the number of
theoretical plates, N: