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Chromatography Essentials
Type of
chromatography
Paper
chromatography

Physical process on
which separation
depends
Partition

Stationary phase

Mobile phase

Water trapped
Solvent ( liquid)
between cellulose
fibres on paper
( liquid)
Thin layer
Adsorption
Solid alumina or
Solvent ( liquid)
chromatography
silica supported on
a glass or plastic
plate
Gas liquid
Partition
Non volatile or high
Inert gas like
chromatography
boiling point liquid
nitrogen or helium
coated onto small
( carrier gas)
inert particles of
silica
High performance
Partition
Non volatile non
Polar Solvent
liquid
polar liquid coated
(liquid)
chromatography
onto small particles Eg: Methanol/Water
of silica
TLC : normally described as adsorption chromatography but some partitioning
occurs, if some water is present on the stationary phase. ( silica coated on the glass
plate may reabsorb some moisture)
Normally after coating the glass plate with silica or alumina slurry, it is dried in an
oven to ensure all of the water is evaporated off and the layer has no water
molecules.
Both alumina and silica can become rehydrated again. When this happens water
present on the layer also acts as a partitioning stationary phase together with
adsorbing solid phase.
Components in a mixture have different affinities for the stationary phase and for
the mobile phase
Components which have stronger affinity for mobile phase move faster with the
eluent ( solvent in mobile phase)
Components which have stronger affinity for stationary phase, move with the eluent
at a slower rate
Components with stronger affinity for mobile phase have larger solubility in liquid
eluents and greater affinity for gaseous eluents .

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Components with stronger affinity for stationary phase have larger solubility in
liquid stationary phase ( if the stationary phase is a liquid) than in eluent solvent .
If the stationary phase is a solid, they get strongly adsorbed on the solid stationary
phase
The word partition may be loosely used to describe the distribution of the solute
between the stationary phase and the mobile phase and so should not be confused
for the physical process called partition chromatography.
Partition chromatography essentially means that the solute components get
partitioned between the liquid in the stationary phase and the liquid in the mobile
phase. The liquids in stationary phase and in mobile phase should be immiscible.
( You have learnt the principle of partition coefficient : partition of a solute between
two immiscible liquids. Partition coefficient K pc remains constant at a given
temperature for the same set of solute and two immiscible solvents irrespective of
the quantity of solute and volumes of solvents)
Similarly , word adsorption may be used to describe the affinity of a component
for the stationary phase. It may not be actually adsorption. It may be partition. So
you should be able to make correct sense of the term by looking at the physical
states of the stationary phase.
Adsorption necessarily means formation of temporary and weak bonds on the
surface of solid stationary phase.
If the stationary phase is a liquid supported on a solid surface, the physical process
for separation has to be partition as the solute partitions itself between the two
liquids in the stationary and the mobile phases. The mobile phase may be a liquid or
a gas.
If the stationary phase is a solid , then the physical process for separation is
adsorption. In this case some components may form stronger associations with the
stationary phase and tend to stick on to stationary phase rather than moving with
the eluent. Such components will move at a very slow rate.
On the other hand , some components may not form strong associations with the
stationary phase but may dissolve better in the solvent of the mobile phase .These
components will move with the eluent at a faster rate.
The affinity for either of the phases depends on the chemical natures of the
components of the mixture and the chemical natures of both the stationary phase
and the mobile phase.
For example , if the stationary phase is polar, and mobile phase non polar,

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then any polar components of the mixture will interact more strongly with the polar
stationary phase than the non polar components.
This is what will cause the separation to occur.
Polar components will move slowly with the non polar mobile phase solvent, as they
form stronger associations with the polar stationary phase.
Non polar components will move faster with the non polar mobile phase as they
interact better with the non polar mobile phase than with the polar stationary
phase.
If the stationary phase is non polar but mobile phase is polar,
then more polar components will move faster with the polar mobile phase,
and non polar components will interact better with the non polar stationary phase
and hence move at a slow rate.
The same principle is true for all types of chromatography.
Stationary phase is the phase which does not move
Mobile phase moves over the stationary phase.
A daily life example: To be able to understand the principle of chromatography
Lets say you drop some jelly and some biscuit crumbs on the floor. Floor acts as the
stationary phase. Jelly sticks to the floor more strongly than the biscuit crumbs. You
use some water to clear the mess. Water becomes your mobile phase. As you spill
some water , the biscuit crumbs start moving with water easily towards the drain ,
but jelly remains stuck to the floor.
We say jelly is adsorbed on to the stationary phase strongly but biscuit crumbs
interact with mobile phase water better than with stationary phase floor.
If you continue to spill water over and over again, jelly slowly starts to come off the
floor. It may move with water for a short distance and get stuck on the floor again.
This may continue to happen again and again till ultimately jelly goes into the drain.
If you compare biscuit crumbs with jelly, biscuit crumbs move much faster with the
eluant than jelly because the two interacted differently with the stationary and
mobile phases.
Rf values are used to identify components separated by paper chromatography and
TLC.
Rf =

distance moved by the component / distance moved by the solvent front

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This ratio is constant for a solute using the same pair of stationary and mobile
phases at a given temperature. Value of R f will change if either of the above is
changed.
Rf is called the Retardation factor
In GLC and HPLC the components are identified by their Retention times
Retention time : It is the time taken by a component to pass through the column
from the time of injection to the time of elution .
Retention times are recorded as chromatograms showing a set of peaks having a
peak for each of the components.
The area under each peak recorded is proportional to the amount of solute getting
eluted from the column.
For quantitative analysis, THE COMPONENT PEAKS are first identified and then the
area under each peak is calculated using the expression
base x height ,
% of a component =
areas under all peaks

as the peaks are roughly triangular in shape.

100

Area under the peak of component / sum of