M .Pharm Pharmaceutics 1 sem seminar

1. Paper Chromatography
Prepared By :- Arjun S.Dhawale
M.Pharm 1sem Pharmaceutics
Kamla Nehru College of Pharmacy
Butibori , Nagpur .

2. Content :-
1-Introduction .
2-Principle.
3-Migration parameter .
4-Apparatus .
5-Types of paper chromatography .
6-Procedure .
7-Application .

3. Introduction :-
Paper chromatography is an analytical method used to separate coloured chemicals or substances.
A paper chromatography variant, two-dimensional chromatography involves using two solvents and
rotating the paper 90° in between.
This is useful for separating complex mixtures of compounds having similar polarity, for example, amino
acids. The setup has three components. The mobile phase is a solution that travels up the stationary
phase, due to capillary action. The mobile phase is generally a mixture of non-polar organic solvent, while
the stationary phase is polar inorganic solvent water.

4. Principle :-
This technique is a type of partition chromatography in which the substances are distributed between two
liquids .i.e, one is stationary liquid [usually water] which is held in the fibres of the paper and called the
stationary phase . Other is the moving liquid or developing solvent and called mobile phase . The
components of the mixture to be seperated migrate at different rates and appear as spots at different points
on the paper.Cellulose filter paper is often used as the stationary phase in paper chromatography.
Since it is hydrophilic, it is usually covered with thin film of water.
The procedure is often regarded as liquid-liquid chromatography.
The components of the separated migrate at different rates and appear as spots at different points on the
paper.
In this technique, a drop of the test solution is applied as a small spot on a filter paper and the spot is
dried.

5. The paper is kept in a close chamber and the edge of the filter paper is dipped into a solvent called
developing solvent.
As soon as the filter paper gets the liquid through its capillary axis and when it reaches the spot of the test
solution (a mixture of two or more substances), the various substances are moved by solvent system at
various speeds.
When the solvent has moved these substances to a suitable height (15-18 cm) the paper is dried and
various spots are visualized by suitable reagent called visualizing agent.

6. Migration parameter :-
The movements of substances relative to the solvent are expressed in terms of Rf values (retardation
factor or retention factor).
Rf: The R is related to the migration of the solute front relative to the solvent front as :-
RF = Distance travelled by the solute from the origin line
Distance travelled by the solvent from the origin line
R is a function of the partition coefficient. For a given substance it is constant provided the conditions of
chromatographic system are kept constant with respect to temperature, type pf paper, duration and
direction of development, nature and the shape and the size of the wick used (i.e., radial
chromatography), the amount of liquid in the reservoir humidity, etc.
The Rf defines the movement of the substances relative to the solvent front in a given chromatographic
system.

7. The Rf value of a substance depends upon a number of factors which are :-
1-The solvent employed.
2-The medium used for separation, i.e., the quality of the paper in case of paper chromatography
3-The nature of the mixture.
4-The temperature.
5-The size of the vessel in which the operation is carried out.

8. Apparatus :-
1-Beaker
2-Glassrod
3-Paper
4-Lid

9. Types of paper chromatography :-
1-Descending chromatography
.
When the development of the paper is
done by allowing the solvent to travel
down the paper, it is known as descending
technique.

10. 2-Ascending chromatography .
When the development of the paper is
done by allowing the solvent to travel up
the paper, it is known as ascending
technique.

11. 3-Ascending-Descending
Chromatography .
It is the hybrid of the above two
techniques.
In this technique, the upper part of the
ascending chromatography can be folded
over a glass rod allowing the ascending
development to change over into the
descending after crossing the glass rod.

12. 4-Radial paper
Chromatography .
This is also known as circular paper
chromatography.
This makes use of radial development.
In this technique a circular filter paper is
employed.
Then the various materials to be analyzed are
placed at the center.
After drying the spot the paper is fixed
horizontally on the petri-dish possessing the
solvent so that the tongue or the wick.

13. 5-Two-dimensional
chromatography .
In this, a square or rectangular paper is used.
The sample is applied to one of the corners.
The second development is performed at right
angle to the direction of the first run.
This type of chromatography can be carried out
with identical solvent systems in both the
directions or by two solvent systems.

14. Procedure :-
1-Sample preparation .
The mixture (e.g. a mixture of amino acids) to be analysed is dissolved in a suitable
solvent (0.5-3%).
The selected solvent should be volatile for rapid evaporation of the solvent.
2-Paper selection .
Whatman No. 1 chromatographic paper or its equivalent paper is used for analytical
paper chromatography.
3-Sample loading or spotting .
A pencil line is drawn across the paper 10-15cm from the bottom for ascending
paper chromatography and 10-15cm below antisiphon bar for descending.
A drop of solution is placed on the paper at this line by means of a capillary tube,
micro pipette.
Reference compounds are similarly prepared and applied to the paper alongside the
mixture spot.

15. 4-Development or elution .
Paper chromatograms can be developed in a wide variety of development chambers
or tanks.
Small paper strips can be developed in a small jar or wide mouthed screw capped
bottles.
A large rectangular piece (20cm X 20cm) of paper is developed in a large
rectangular glass tank.
The solvent (eluant) is poured into the tank in such a way that the penciled line is
just above the surface of the solvent.
When the solvent reaches almost the top of the paper, the paper is removed from the
tank and the solvent is allowed to evaporate in air until the paper is dry.

16. 5-Detection of spot location or visualization .
If the separated components or solutes are colored, they canbe easily seen in daylight
under naked eye.
If the components are colorless, a physical or chemical property must be employed to
reveal or locate their positions.
Widely used visualization techniques are fluorescence and spray reagent but conc.
sulphuric acid is not practical for paper chromatography because the paper is charred.
Spray reagents are chemical reagents, which react with functional groups to produce a
colored spot.
For e.g., after the development, the paper is sprayed with a solution of ninhydrin
reagent then heated for 10 minutes at 1100 C to form a coloured compound with each of
the amino acids.

17. Applications of paper chromatography .
1-Qualitative analysis :
Involves the identification of compounds present in the mixture.
Identification involves the use of Rf value based on Rf of standard compound.
2-Quantitative analysis :
It is done in the paper or after the removal of the component from the paper.
The latter is generally preferred – the component is cut from the paper, extracted by a
suitable solvent, measured by colorimeter or UV-Vis spectrophotometer.
Alternatively, the extracted solution is evaporated in the vacuum to remove solvent,
Thus, obtained residue is weighed.

18. 3-Preparative paper chromatography .
Operates with large amount (gram quantity) of substances to yield substances enough
for further work in the laboratory.
Practically, it is done in Whatman No.3 paper. The sample is streaked.
The separated bands are cut, extracted with suitable solvent and filtered.
The filtrate is evaporated off in vacuum to yield the residue of the component.
4-Specific application .
Includes the separation of many organic and biochemical products.
For example, it has been utilized in the determination of indole in whole urine and in
the study of barbiturates, antibiotics, hormones, and amino acids, among others.

19. Thank you