principle, requirements, method and applications of paper chromatography

1. PAPER
CHROMATOGRAPHY

2. PAPER CHROMATOGRAPHY -
INTRODUCTION
Paper chromatography is an analytical method used to separate colored
chemicals or substances.
It is primarily used as a teaching tool, having been replaced by other
chromatography methods, such as thin-layer chromatography.
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.

3. The mobile phase is a solution that travels up the stationary phase, due
to capillary action.
The mobile phase is generally mixture of non-polar organic solvent,
while the stationary phase is polar inorganic solvent water.
Here paper is used to support the stationary phase, water.
Polar water molecules are held inside the void space of cellulose
network of the host paper.
Difference between TLC and paper chromatography is that stationary
phase in TLC is a layer of adsorbent (usually silica gel, or aluminium
oxide), and stationary phase in paper chromatography is water.

5. WHATMANN FILTER PAPER
It is used as stationary phase in paper
chromatography

11. TYPES OF PAPER CHROMATOGRAPHY
1. Descending
2. Ascending
3. Ascending-descending
4. Circular chromatography
5. Two-dimensional

12. DESCENDING PAPER CHROMATOGRAPHY
- Development of the chromatogram is done by
allowing the solvent to travel down the paper.
- Here, mobile phase is placed in solvent holder
at the top.
- The spot is kept at the top and solvent flows
down the paper from above.

14. ASCENDING PAPER CHROMATOGRAPHY
- Here the solvent travels up the chromatographic
paper.
- Both descending and ascending paper
chromatography are used for the separation of
organic and inorganic substances.
- The sample and solvent move upward.

16. Ascending-descending
- This is the hybrid of both of the above
techniques.
- The upper part of ascending
chromatography can be folded over a rod in
order to allow the paper to become
descending after crossing the rod.

18. Circular chromatography
- A circular filter paper is taken and the sample is
deposited at the center of the paper.
- After drying the spot, the filter paper is tied
horizontally on a Petri dish containing solvent, so
that the wick of the paper is dipped in the solvent.
- The solvent rises through the wick and the
components are separated into concentric rings.

20. RF VALUE - EXAMPLE
If a compound travels 9.9 cm and the solvent front
travels 12.7 cm, then
Rƒ value = 9.9/12.7 = 0.779 or 0.78.
Rƒ value depends on temperature and the solvent
used in experiment, so several solvents offer
several Rƒ values for the same mixture of
compound.

21. TWO DIMENSIONAL PAPER
CHROMATOGRAPHY
- In this technique a square or rectangular
paper is used.
- Here the sample is applied to one of the
corners and development is performed at a
right angle to the direction of the first run.

23. PRINCIPLE
The principle involved can be partition chromatography.
Partition chromatography because the substances are partitioned or
distributed between liquid phases.
The two phases are water held in pores of the filter paper and the other
phase is a mobile phase which passes through the paper.
When the mobile phase moves, the separation of the mixture takes place.
The compounds in the mixture separate themselves based on the
differences in their affinity towards stationary and mobile phase solvents
under the capillary action of pores in the paper.

26. PROCEDURE
1. Selecting a suitable type of development:
Usually ascending type or radial paper chromatography is
used as they are easy to perform.
Also, it is easy to handle, the chromatogram obtained is
faster and the process is less time-consuming.
1. Selecting a suitable filter paper:
Selection of filter paper is done based on the size of the
pores, and the sample quality.

27. 3. Prepare the sample:
Sample preparation includes the dissolution of
the sample in a suitable solvent used in making
the mobile phase.
4. Spot the sample on the paper:
Samples should be spotted at a proper position
on the paper by using a capillary tube.

28. 5. Chromatogram development:
Chromatogram development is spotted by immersing the
paper in the mobile phase. Due to the capillary action of
paper, the mobile phase moves over the sample on the paper.
6. Paper drying and compound detection:
Once the chromatogram is developed, the paper is dried
using an air drier.
Also, detecting solution can be sprayed on the chromatogram
developed paper and dried to identify the sample
chromatogram spots.

29. Visualizing methods
1.UV chamber/cabinet
2.Iodine chamber
3.Spraying agents

30. UV chamber or cabinet

31. TLC plate under UV light

32. Iodine chamber with iodine crystals

33. Spraying by sprayer

34. Specific spray reagents

35. Calculation of Rf Value

36. Calculation of Rf Value
Rf = Retention or retardation factor
Distance travelled by the solute
Rf = …………………………………………………….
Distance travelled by solvent

37. Maximum value of Rf = 1
The retention factor (Rƒ) may be defined as the ratio of
the distance traveled by the solute to the distance
traveled by the solvent.
● If Rƒ value of a solution is zero, the solute remains in
the stationary phase and thus it is immobile.
● If Rƒ value = 1 then the solute has no affinity for the
stationary phase and travels with the solvent front.

38. Example
If distance travelled by the solute = 6 cm
And distance travelled by solvent - 8 cm
Then Distance travelled by the solute
Rf = …………………………………………………….= 6/8 = 0.75
Distance travelled by solvent

40. APPLICATIONS
● To study the process of fermentation and ripening.
● To check the purity of pharmaceuticals.
● To inspect cosmetics.
● To detect the adulterants.
● To detect the contaminants in drinks and foods.
● To examine the reaction mixtures in biochemical
laboratories.
● To determine dopes and drugs in humans and animals.

43. TAPAN KUMAR MAHATO
tapan.mahato@gmail.com