Paper chromatography is a planar chromatography technique using paper as a stationary phase to separate compounds based on their partitioning between stationary and mobile phases. It involves methodologies such as sample loading, solvent development, and spraying, with various solvent systems utilized to enhance separation. While it offers advantages of simplicity and cost-effectiveness, it has limitations like time consumption and inability to use certain detection methods.

1. Techniques of separation
Module : Chromatography
Topic : Paper Chromatography
By
Dr. M. T. Bachute
Dept. of Chemistry
K.B.P. Mahavidyalaya, Pandharpur

2. Paper Chromatography
Paper chromatography is a type of Planar
chromatography in which paper is the support
for stationary phase.
It is a partition chromatography.
Stationary Phase : Liquid, deposited in network
of cellulose fibers.
Mobile Phase : Liquid, solvent(single or
mixture)
It is a liquid – liquid chromatography

3. Principle
It is based on the principle of partition of a compound
between two liquid phases.
The water hold in cellulose fibers of the paper acts as the
stationary phase and acts as one of the solvents (liquids).
When a non–aqueous mobile phase moves along the paper,
the components of the loaded mixture distribute
themselves between two phases in the ratio characteristic
of their distribution coefficients.
The component which is more soluble in stationary phase
moves slowly while the component which is more soluble in
mobile phase moves fast.
As a result of this differential movement, components get
separated.
Distribution coefficient = (solute in stationary phase) ÷ (solute in mobile phase)

4. Important Aspects of Chromatography
1. Filter Paper
2. Solvent System
3. Spraying reagents

5. Filter Paper
• Usually Whatman paper No. 1 is used.
Characters : Uniform flow rate
Grain size and texture
• Some times the papers are acid
washed to remove traces of
impurities which may affect
the analysis.
Strip of whatman No. 1 paper

6. Solvent system
Solvent is prepared by saturating organic solvent
with water.
But several organic liquids incorporate only small
amount of water. In such binary solvent system
polar components of the mixture fail to
separate.
In such cases a third liquid like acid(HCl, AcOH,
HCOOH) or base(NH3 or pyridine) or a
complexing agent is often added to retain more
water in organic solvents.
Hence, generally three component solvent
systems are used

7. Some important solvent systems
Compounds Solvent system Spraying reagent
1. Amino acids Butanol: Pyridine:Water
(33 :33 :33)
Ninhydrin
Methanol:Pyridine:Water
(25 : 12 :63)
Butanol : AcOH : Water
(40 : 19 50)
Phenol : Water
(80 : 20)
2. Carbhydrates(mono , di) Butanol : AcOH : Water
Butanol:
Pyridine:Water:AcOH
(8:8:4:1)
Ammoniacal AgNO3
Alkaline KMnO4
3. Chlorophils and
carotenoids
Propanol : Pet. Ether
Chloroform : Pet. ether
Self
4. Metal ions Ethanol: 5M HCl (90 : 10) Rubeanic acid, NH3

8. Spraying reagents
• Coloured components do not require spraying
reagent, they are visible.
• Colourless components are located by
spraying with specific reagents. These
reagents chemically react with the
components and develop colour in the region
where they are present.

9. Methodology
1. Sample loading
Preparation of sample solution :
1-2% solution is prepared in suitable
solvent.
A reference line at 2cm . Distance
from the edge of the paper is drawn
and points of application are marked.
The solution of mixture under test is
spotted either as a fine line or fine
spot. Spotting is repeated twice or
thrice. Reference Line .

10. Methodology
2. Developing
Saturation of the paper strip: The sample loaded strip is
hanged for some time in the chromatographic jar
containing the solvent but without dipping in the
solvent.
The strip is developed by running the solvent through the
paper.
Depending on the direction of flow, there are two types
paper chromatography.
A. Ascending paper chromatography
B. Descending paper chromatography

11. Ascending P. C.
Solvent moves upward(ascends) from the
bottom to top by capillary action. The paper
strip is placed vertically in the solvent keeping
the reference line above the solvent level.
Pencil
Filter
Paper
Ink
Mark
Tape – Label with marker

12. Descending P. C.
• Solvent moves downward(descends) from the
top to bottom. The paper strip is hanged
vertically downward from the trough
containing solvent, keeping the spotted end
at the top.
The flow of solvent is faster than that in
ascending P.C.
But A.P.C. is preferred than D.P.C. due to
simplicity of set up.

13. Radial or Circular chromatography
The plane of paper is kept horizontal and solvent
moves laterally(toward the periphery) from
the center.

14. Methodology
3. Spraying : After movement of the solvent to
expected distance(15 to 18 cm), paper is
removed, dried and sprayed with suitable
reagent.

15. Descending paper chromatography

16. Rate of Flow : Rf value
Rf value : It is the ratio of distance travelled by
the solute to the distance travelled by
the solvent front. i.e.
Distance moved by solute
Rf =
Distance moved by the solvent front
Rf value …….
• characterises the distance moved by the solute in the direction of solvent
flow
• under standard conditions it closely reflects the distribution coefficient
and it is characteristic of that compound

17. Rf value
Factors affecting Rf value
1. Quality and dimension of the paper.
2. Solvent system, its flow rate and direction of
flow.
3. Temperature of the environment
4. Size of the vessel in which chromatogram is
developed.
If these factors are kept constant, Rf values of
known and unknown compounds can be
compared.

18. Applications of Paper Chromatography
1. Separation of amino acids, peptides,
alkaloids, sugars, lipids etc in biological
samples.
2. To limited extent as a preparative method.
3. To study the structure and amino acid
composition of proteins
4. Analysis of blood, hemoglobin, urine etc
which is of great diagnostic value.

19. Advantages
1. Method is simple, inexpensive and sensitive.
2. Separations can be carried out under
ordinary lab. conditions.

20. Disadvantages
1. Time required is large.
2. It can not be used as a preparative method.
3. Heating or corrosive reagents can not be
used for detection.

21. Advantages of TLC over PC
1. Versatility in the use of stationary phase.
It offers choice between wide range of adsorbents to suite the
separation of variety of components.
2. Adsorbent layer of variable thickness(upto 5mm)can be obtained.
Higher sample load is possible.(higher load on paper not possible)
3. Method is very rapid & most of the separations can be completed
within an hour.
(PC requires 18 to 24 hrs.)
4. Separated spots are very compact, hence detection at lower
concentration is possible.
(In PC spots are diffused)
5. Higher temp. and corrosive sprays can be used to detect unknown
solutes.( higher temp and corrosive sprays can not be used with
paper.)
6. Since it is simple, rapid and inexpensive, it can be used to check
purity of synthesised or isolated chemicals.

22. Disadvantages of TLC
1. Rf values are not reproducible. Rf value is
affected by thickness of the layer below
0.2mm and also by uneven thickness of the
layer.