Paper chromatography is a technique used to separate mixtures by distributing components between a stationary and mobile phase. In paper chromatography, specially designed paper acts as the stationary phase, while a solvent moves along the paper as the mobile phase. Separation occurs as components partition differently between the bound water in the paper and the mobile phase solvent. The distance traveled by each component is used to calculate its retention factor (Rf) value for identification. Various detection methods can be used to visualize separated components on the paper. Paper chromatography is a simple, efficient, and inexpensive method to analyze mixtures in many applications.

1. Paper Chromatography

2. CHROMATOGRAPHY
The name chromatography means color
writing (In Greek Chroma means colour
Graphy means writing).
Chromatography is a physical/automated
method of separation in which the
components to be separated are
distributed between two phases i.e.
stationary phase and mobile phase moves
in a definite direction.

3. PAPER CHROMATOGRAPHY
• “As the technique in which the analysis of unknown
substances is carried out mainly by the flow of
solvents on specially designed paper.”
• In PC, paper with controlled texture & thickness is
used.
• Paper made of cellulose, a hydroxylated
polysaccharide have more affinity for water & other
polar solvents.
• Tightly bound water is the actual stationary phase &
as the mobile phase passes over the surface of the
paper, the solute distributes themselves between
the bound water & the mobile phase.
• Mechanism involves in PC is partition.

5. Partition occurs between the mobile phase and the
stationary aqueous phase bound by the cellulose.
The isolation depends on partition coefficient of the
solute.
( )
( )
c stationary
K
c mobile


6. Stationary Phase in PC
• Thin papers: No. 4, 54, 540 (fast flow), No. 7 & 1
(medium flow), No. 2 & 20 (slow flow)
• Thick papers: No. 17 & 31 (fast ).
• Modified papers:
a) Carboxyl papers- For amines & AAs.
b) Acetylated papers- for lipophilic subs like
steroids, insecticides, pigments.
c) Kieselguhr papers, Alumina papers- for low
polarity subs like amines, fatty acids, steroids, TGs,
vitamins, pesticides.

7. Mobile phase in PC
Commonly used (increasing polarity) :
n-Hexane,
Cyclohexane,
CCl4
Benzene,
Toluene,
Trichloroethylene,
Diethyl ether,
CHCl3,
Ethylacetate,
n-butanol,
n-propanol,
Acetone,
Ethanol,
Methanol,
Water.

8. General Procedure
1- Choice of paper and solvent to be used.
2- Desalting of sample.
3- Application of the sample.
4- Equilibration of paper.
5- Development.
6- Detection.
7- Identification of substances.

9. General Procedure
• A small spot of the analyte is applied on one
end of the paper and dried
• Then placed in the closed chamber and
spotted edge of the paper is dipped into the
developing solvent such that spot remain well
above the surface of the solvent
• The solvent moves upward through capillary
action, passes the spot and runs up the paper.

11. • The separation is due to differential migration
of solutes due to difference in partition
coefficients.
• Paper is dried after the solvent front has
moved almost the height of the paper and the
various spots are visualized if coloured or
visualizing agents are used
• Then Rf value is calculated
R 𝑓 =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
distance travelled by solvent

12. Spot development

13. Paper development
There are four main techniques, which may be
employed for the development of paper
Chromatograms.
1) Ascending techniques
2) Descending techniques
3) Radial development
4) Two-dimensional chromatography

14. Techniques of development with various flow
directions
Ascending development
Descending development
Radial development

15. PAPER CHROMATOGRAPHY
• Ascending, Descending & Radial

16. Two-dimensional chromatography:
• When large numbers of substances are to be separated
on a single chromatogram.
• Development in a direction perpendicular to the first,
and with a solvent system different from that used
initially is often necessary.
• The sample is applied on one corner of a square piece
of paper and after development with the first solvent,
the paper is dried , rotated 90o and developed in the
second direction.
• Usually, different types of solvents systems are used in
each direction. It is essential that the first solvent be
completely volatile.

19. Locating the compounds:
• Strip is removed when the solvent has
migrated over most of the available space. The
distance to which the solvent has run is
marked. In most cases, the completed
Chromatogram is colourless with no indication
of the presence of any compounds. Such a
chromatogram is said as “Undeveloped” for
locating the various compounds.

20. Identifying the compounds:
• The ratio of the distance travelled by a
component to that travelled by the solvent
front, both measured from the marked point of
the application of the mixture, is called the
“Retention factor (Rf)” value for that
component.

21. Spraying Regent/Visualizing Agents
Alkaloids: Dragendorff’s reagent-Orange or
orange yellow spots.
Cardiac glycosides: Antimony trichloride
Sugar: Aniline phthalate
Amino acids: Ninhydrin- blue color
Aldehydes & Ketones– 2,4-DNPH
(Dinitrophenylhydrazine) spray in methanol
and sulphuric acid results in orange or
yellow spots

22. ANALYSIS ON PC
Two methods-
1. Evaluation of substances on the paper directly- visual
comparison of spots, measurement of area of spot,
radiotracer analysis.
2. Removal of substances from the paper –cut the
developed spot dissolve completely dissolve in suitable
solvent filter and analyze by suitable method/s.

23. ADVANTAGES
• Equipment/Process is very simple.
• Has high efficiency of separation.
• Separation can be possible for small amount
sample

24. Applications:
By using this technique
1) To check the control of purity of pharmaceuticals,
2) To the detection of adulterants,
3) To detect the contaminants in foods and drinks,
4) To the study of ripening and fermentation,
5) To the detection of drugs and dopes in animals & humans
6) To the analysis of cosmetics
7) To the analysis of the reaction mixtures in biochemical
labs.
8) Study of food colours by PC
9) To determine the amino acids -by 2D method.
10) Separation of mixtures of sugar by PC
11) Separation of a mixtures of dyes by PC

25. References
• Pharmaceutical Analysis-II, Instrumental
Methods by P.C. Kamboj
• Instrument methods of analysis by Scoog and
West
• Text book of Pharmaceutical Analysis by Dr.
S.R.Sankar

27. THANK YOU