2 Paper Chromatography

Paper Chromatography
• This is a powerful method of liquid-liquid Chromatography, used

widely to separate free amino acids (produced as result of hydrolysis
of protein material), sugars, peptides and sugar derivatives.
• Let us consider the e.g. of separation of amino acids.
• A thin pencil line is drawn near one edge of a rectangular piece of
filter paper and parallel to it.
• The solid mixture present in solution is now applied as a small drop
from a fine glass syringe along the pencil line and near one vertical
edge of the paper If the problem is to identify the amino acids in a
mixture, then put small drops of separate solution of pure amino acids
(which we expect to be present in the mixture) at intervals along the
pencil line a bit away from mixture drop.
A B
• The filter paper is now hung in a glass tank, which contains the developing
solvent and the bottom edge of the filter paper is so positioned in the solvent
that the pencil line is just clear of it. The tank is sealed with a lid to prevent the
evaporation of the solvent.
• As the solvent travels up (due to capillary action) through the paper it carries
the components in the mixture as well as the pure substances to different
heights. When the solvent has almost reached the top, the paper is removed
from the tank and is allowed to dry. Since, amino acids are colourless thus
whole of the paper is sprayed by a solution of ninhydrin in propanone and
paper is dried in an oven set at about 373ᵒ K.
• The individual amino acids show up as different colour spots.
Under carefully controlled conditions it is possible to characterize a particular
compound separated from the mixture by its Retention factor value.
• It is clear from this formula that value will always be less than unity.
Two way chromatography
• In some cases the use of one solvent may fail to separate
two or more of the components present in a mixture i.e.
their value is quite close to each another then under such
conditions another solvent is used and this time the same
paper is dipped by rotating it through . The logic behind it
is that it is impossible that two or more components have
identical or close values in different solvents This is
called two way chromatography.
Thin-layer chromatography
In this chromatography the filter paper of paper
chromatography is replaced by a thin layer of an inert
adsorbent (like cellulose, alumina or silica gel) spread
over a square or rectangular plate of glass or plastic
This method is basically used to identify the
components in a small sample and to separate them.
1. A slurry of silica gel is made in trichloro methane contained
in an ordinary screw cap bottle.
2. A previously cleaned microscope slide is immersed in the
Process to slurry and then carefully withdrawn using a pair of tweezers.
carry out 3. The coated glass plate is now allowed to dry in an upright
position and is then ready for use.
thin layer
4. At a distance of about 2.0 – 2.5 cm from the bottom of plate,
chromatography a horizontal line is drawn.
5. With the help of a syringe or micropipette, spots of sample
solution and standards are applied.
6. Plate is kept in a chamber.
7. Chromatogram is developed by immersing the plate in a
developing solvent to a depth of 0.5 cm.
8. Development is allowed to proceed until the solvent
front has travelled the required distance i.e. 10 – 15 cm.
Process to 9. Plate is removed from the chamber and the height of
solvent front is marked with a pointed object.
carry out
10. Plate is then allowed to dry in oven.
thin layer 11. The position of separated solutes can be located by
chromatography various methods.
(contd.) 12. Colored substances can be detected directly by seeing
to it.
13. If colorless then, either by spraying with appropriate
reagent to give colored compound or by using their
fluorescence property if any by using U.V. light.
Depending upon the nature of substances to be
studied we can use different types of thin film
e.g., silica gel, alumina or sephadex.
Advantages of The thin film is more compact than filter paper,

thin layer so spots are much smaller and concentrated and
easy to identify even at low concentration.
over paper
Chromatography Process is quite rapid as flow of solvent is smooth
and faster.
It is useful in identifying and isolating amino

acids, nucleotides, triglycerides, other lipids,
sterols, sugars and sugar derivatives.
Gas Chromatography
It is a technique by which a mixture is separated into its constituents by a moving

gas phase passing over the stationary absorbent which may be either a liquid or
solid.
In this chromatography if adsorption is taking place i.e. stationary phase is solid

then technique is called gas-solid chromatography (GSC), while if stationary
phase is liquid and portioning process is responsible for separation then the
technique is called (GLC).
Instrumentation:
A gas-chromatograph consists of:
• A supply of a carrier gas (He, Ar or N2) from a high pressure cylinder
having a pressure regulator and flow meters.
• A sample injection system.
• Packing materials is packed column or open tabular or capillary column
are as follows:
a) In GSC – activated carbon, silica gel or alumina.
b) In GLC – porous materials like glass beads, ground fire bricks, finely
divided celite or polymers like tenax GC, poropak, chromosorb, etc.
• The detector is situated at the exit of the separation column. Which senses
and measures the small amount of the separated components present in the
carrier-gas leaving the column.
Commonly used detectors are thermal conductivity detector, wheatstone

bridge circuit and flame ionization detector.
Note: The temperature of detector compartment should be sufficiently high to

avoid-condensation of sample vapors, but not so high that it causes sample
decomposition.
GLC is used to separate mixture of gases, liquids and volatile solids. A
systematic GLC instrument is as follows:
The stationary phase – A non-volatile long chain alkane deposited on an inert
material is packed into a narrow and long column 20 m long and 4 mm wide bend
into a shape of a U-tube.
Mobile phase – A gas which does not interact with any of the components that have
to be separated also known as carrier gas mostly argon or nitrogen is used.
The progress of gas through the column is controlled by a pressure controller or say
valve and monitored by a flow meter.
The mixture to be separated is injected directly onto the top of the column through
the self-sealing septum cap by means of hypodermic syringe. The partition of
components in the mixture occur to different extent between the stationary and
mobile phases and they thus emerge from the column at different time intervals.
When dealing with mixtures of components having different degrees of
volatility then provision is made for variation of temperature in the column
which is thermostatically controlled.
One of the most common means to detect the emerging components is

measuring their thermal conductivities which is done by a device called
katharometer.
The time taken by a component to emerge out of the column is called the
retention time. For a particular carrier gas and flow rate particular stationary
phase and column temperature retention time can be used to identify a
particular component.
•A gas chromatogram of a mixture of six
components.
•The retention time for Hex-1ene = (B-A) min.
•The retention time for Heptane = (C-A) min.
•The area under the peak is directly proportional

to the amount of the component and if we have
carried out the experiment with a known amount
of any pure component then we can find out its
concentration in the mixture from the
chromatogram.
•Since two compounds can have same retention

time so we can identify them by using two or
three different types of stationary phases.
Applications
1. Analysis of natural gases.
2. To analyze gasoline plant samples.
3. Analysis of refinery gases.
4. To identify synthetic rubber and plastic intermediates.
5. To detect trace atmospheric constituents.
6. Actually, separation of close boiling benzene (80.1 ℃) and cyclo hexane
(80.8℃) is not possible by distillation but can be carried out by GLC.
7. By using GLC the ethyl alcohol content in blood can be determined with high
accuracy using glycol column.

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Paper Chromatography

• This is a powerful method of liquid-liquid Chromatography, used

Advantages of The thin film is more compact than filter paper,

It is useful in identifying and isolating amino

It is a technique by which a mixture is separated into its constituents by a moving

In this chromatography if adsorption is taking place i.e. stationary phase is solid

Commonly used detectors are thermal conductivity detector, wheatstone

Note: The temperature of detector compartment should be sufficiently high to

One of the most common means to detect the emerging components is

•The area under the peak is directly proportional

•Since two compounds can have same retention

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