Paper chromatography is a method used to separate components of a mixture based on how they partition between a stationary phase, typically cellulose paper, and a mobile phase, typically a solvent. Key aspects of paper chromatography include choosing an appropriate paper and solvent based on the sample's properties, applying the sample as a spot on the paper, developing the chromatogram by allowing the solvent to migrate up the paper via capillary action, and detecting separated components using physical or chemical methods. Results are represented by each component's retention factor (Rf) value, which indicates its relative migration distance compared to the solvent front. Variations in development technique include ascending, descending, and two-dimensional paper chromatography. Paper chromatography has wide applications in chemistry, biology

1. Presented by:
Vambhurkar Ganesh B.
M.Pharm 1st Year (Pharmaceutics)

2. Introduction:
The term chromatography (In Greek ,kromatous-colour and Graphos-Written)
meaning writing.
-It is the method in which the component of a mixture are separated on an
adsorbents.
-It is method used primarily for the separation of components of a sample, in
which the components are distributed between two phases , one of which is
stationary while the other moves.
-The stationary phase may be solid or liquid supported on a solid or a gel ,and
may be packed in a column ,sprayed as a layer or distributed as a film. The
mobile phase may be gaseous liquid

3. Classification:
Partition chromatography :
-This involve liquid or gas as mobile phase and another liquid as a
stationary phase.
Adsorption chromatography :
-These involve liquid or gas as mobile phase and adsorbent solid as
stationary phase .

4. Principle:
-The principle involved in separation by paper chromatography
is largely by partition coefficient phenomenon.
-Separation of component depends on both their solubility in
the mobile phase and their differential affinity to the mobile
phase and stationary phase .

5. Operational technique:
Choice of filter paper :
-Whatman filter paper are used as chromatography .
-In general this paper contains 98-99% of α-cellulose .
-There are various grade and types of paper available for
separation of sample.
Factors that governs the choice of paper:
-Nature of sample and solvent used.
-Based on quantitative or qualitative analysis.
-Based on thickness of paper.

6. Preparation of Paper :
-Cut the paper into desired shape and size depending upon work
to be carried out.
-The starting line is marked on the paper with an ordinary pencil
2cm from the bottom edge.
-On the starting line marks are made 2cm apart from each other.

7. Application of sample:
- The sample mixture to be separated is applied as a small
spot on the origin line.
-The spot is dried on the filter paper and is placed in
developing chamber.
-Micropipette or glass capillary is used for sample
application.

8. Solvents:
A number of solvents can be used in the paper
chromatography. The solvent selection depends upon
nature of substance to be separated.
Eg: Ethyl alcohol Benzene
N-Hexane
Toluene
Water
Chloroform
These solvents are used indifferent ratio with different
mixture…

9. Chromatographic Chamber:
-The chromatographic chamber is made up of many
materials like glass, plastic or stainless steel.
-Glass tanks are preferred most .They are available in
various dimensional size depending upon paper length and
development type .
-The chamber atmosphere should be saturated with solvent
vapour.

10. Development of Chromatogram:
-The paper is dipped in solvent in such a manner that the
spot will not deep completely into the solvent .
-The solvent will rise up by capillary action .It is allowed to
run 2/3rd of paper height for better and efficient result.
-After development is complete paper is taken out of the
chamber carefully.

11. Drying of chromatogram:
-The chromatogram is dried after its development .
-They are dried by cold or hot air depending on volatility of
solvent .A simple hair dryer is a convenient device to dry
chromatograms.

12. Location of Spot:
-If the substance are coloured they are visually detected
easily .
-But for colourless substance .Physical and chemical
method are used to detect spot.
.

13. Physical method:
-In these method observation are done under uv light
,detection of fluorescence and radioisotope measurement

14. Chemical method:
-In this method chemical reagent is used to develop the
colour .
-Amino Acid-Ninhydrin Reagent .
-Alkaloids –Dragendroff’s Reagent.

15. Rf value:
In paper chromatography the results are represented by Rf
value which represent the movement or migration of solute
relative to the solvent front .

16. Development technique:
- Ascending chromatography
- Descending chromatography
- Ascending – Descending chromatography
- Radial chromatography
- Two dimensional chromatography

17. Ascending Chromatography:
In these case the solvent migrates upward by capillary action.

18. Descending Chromatography:
-In this method solvents moves from top to bottom so it is
called descending chromatography.

20. Ascending –Descending
Chromatography:
A hybrid of above two techniques is called ascending-
descending chromatography.
Initial chromatography is performed ,often crossing the
glass rod changes to descending.

22. Radial Chromatography:
- These is rarely used method; in this case a circular piece of
paper is taken which has a wick cut parallel to the radius
from the edge to centre .
-The sample is applied at the centre of the paper. The paper
is then laid on the edge of circular disc with wick dipped
into the solvent at the bottom of the dish

24. Two Dimensional Chromatography:
-In this method a square paper is taken the sample is applied
to the one of the corner .Using solvent system the first
development is carried as ascending method .
-The paper is taken out dried and second development is
performed at right angle to the first dimensional
development.

26. Application of the paper
chromatography:
-Used in the separation of various organic mixture.
-Used for both qualitative and quantitative analysis.
-Used in almost all area to solve complicated problem in
chemistry, biology, biochemistry.