The document provides a comprehensive overview of paper chromatography, covering its principles, types, methodologies, and applications. It explains the importance of factors like the choice of filter paper, development solvent, and spotting techniques in achieving effective separation of substances. Additionally, it highlights the various techniques used in paper chromatography, such as ascending, descending, and two-dimensional chromatography, as well as their practical applications in analyzing organic and biochemical products.

1. REPRESENTED BY :-
PARMESHWAR M.
PATHADE

2. TABLE OF
CONTENTS
05
04
02
01
03
INTRODUCTION TO
PAPER
CHR0MATOGRAPHY
PRINCIPLE OF PAPER
CHROMATOGRAPHY
MIGRATION
PARAMETERS
TYPES OF PAPER
CHROMATOGRAPHY
METHODOLOGY
AND APPLICATION

3. INTRODUCTION TO PAPER
CHROMATOGRAPHY
 Cellulose filter paper is often used as the
statioary phase in the paper chromatography.
Since it is hydrophillic, it is usually covered with
thin film of water. The procedure is often
regarded as liquid-liquid cromatography
 Other liquids can be encorporated in place of
water, thus provides different type of stationary
phase. Eg. Paper treated with silicone or
paraffin oil permits reverse phase-paper
chromatography, in which mobile phase is a
polar solvent.
 There are some commercially available papers
that contain an adsorbent or an ion-exchange
resin, thus permmits adsorption and ion-
exhange paper chromatography.

4. PRINCIPLE OF CHROMATOGRAPHY
This is type of partition chromatography in which the
substance are distributed between two liquids that is
one is the stationary liquid (usually water) which is held
in the fibres of paper and called the stationary phase;
the other is the moving liquid or developing solvent and
called the mobile phase. The components of mixture to
be separated migrates at different rates as its solubility
between two phases and appear as spot 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. The
paper is kept in close chamber and the edge of filter is
dipped into a solvent called as developing solvent. As
soon as filter paper gets liquids via capillary action and
reaches to the spot of the test solution then various
substances are moved by solvent with various speeds.
When solvent move up to suitable height (15-18) the
paper is dried and various spot are visualised by
suitable reagent called visualising reagent.

5. MIGRATION PARAMETERS
1) RF VALUE(RETENSION FACTOR) :- It is ratio of the solute’s
distance travelled to solvent’s distance travelled.
It is constant for a given substance, provided the conditions of
chromatographic system are kept constant with respect to
tempreture, type of 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 of of a substance depends upon a number of factors which are:
i. The solvent employed
ii. The medium used for separation i.e., the quality of paper
chromatography
iii. The nature of mixture
iv. The tempreture
v. Size of vessel in which operation has been carried out
It is possible to compare the Rf Values of different substances keeping
above factor constant

6. Distance travelled by the solute from the origine line
Rf = Distance travelled by the solvent from the origine line

7. 2) Rx VALUE :- In some cases, the solvent front
runs off the end of filter paper, the movement of
substance in such cases is expressed as Rx
Distance travelled by the substance from the origine
line
Rx VALUE=
Distance travelled by the standard substance x from the
origine line

8. 3) RM VALUE :- The term RM value is
additive and it is composed of the partial RM
value of the individual functional groups or
other grouping atoms in the molecules.
RM VALUE = log (1/ Rf -1)

9. TYPES OF PAPER
CHROMATOGRAPY
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.
The advantage of descending
chromatography is that the development
can be continued indefinitely even
though the solvents runs off at the other
end of the paper

10. 2) ASCENDING HROMATOGRAPHY :- When the
development of the paper is done by allowing the
solvent to travel up the, it is known as ascending
chromatography.
In ascending chromatography, the mobile phase is
placed in a suitable container at the bottom of the
chamber or in the bottom itelf. The sample are applied a
few centimeters from the bottom edge of the paper

11. ASCENDING
CHROMATOGRAPHY
3) Ascending-descending chromatography:- It is
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

12. Ascending-Descending
chromatography
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 material to be analysed are placed
at the centre. After drying the spot the paper is fixed horizontally
on the petri-dish processing the solvent so that wick of paper
dips into the solvent. Cover the paper by means of petri-dish
cover. The solvent rises through the wick. When solvent front
has moved through a sufficient large distance, the components
get separated in the form of concentric circular zones

13. 5) TWO DIAMENTIONAL 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 first run. This type of chromatography can
be carried out with identical solvent system in both

14. This technique can be understood by spotting the sample at the lower
corner of a rectangular sheet of filter paper so that the spot is situated
above the surface of the developing solvent in the trough. The dried
paper is then kept with its edge in the solvent and developed by either
ascending or descending technique. When the solvent reaches the
opposite edge of the paper, it is removed from the tank and dried. The
solvent system is now changed to a second liquid. The filter paper is now
rotated through 90° so that the edge having the series of spots is now at
the bottom, just above the solvent trough and the chromatogram is run
as before. The chromatogram is now having spot of the solute scattered

15. METHODOLOGY
1) CHOICE OF THE PROPER CHROMATOGRAPHIC
TECHNIQUE :- The first job is to select the mode of
paper chromatographic technique, i.e., ascending,
descending, ascending-descending, radial or two-
dimensional technique. The choice of technique
depends upon the nature of the substances to be
separated.
2) CHOICE OF THE FILTER PAPER :- The filter paper
plays an important role in the success of paper
chromatography. The choice of paper is dependent on
the type of problem under investigation. The prime
factors, that govern the choice, are as follows:
(i) Whether the paper is being used for quantitative or
qualitative analysis;
(ii) Whether it is used for analytical or preparative

16. (iii) Whether the substances used are hydrophilic or
lipophilic neutral or charged species.
Various types of Whatmann chromatography papers are
available
Coarser/ faster Whatmann 31 ET Used when Rf values
are wide apart
Slow Whatmann 20
schleicher, Schull 3045
a, Edrol 208, Machery
Nagel 261.
Used when Rf values
are close
Heavy Schleicher, Schull
2071.
Used for preaparative
purpose

17. Rate of flow
fast Medium slow
No.4 No.7 No.2
Thin papers No.54 No.1 No. 20
No. 540
Thick papers No.31 No.3
No.17 No. 3MM
Characteristics of whatmann chromatography paper
are summarised in below table

18. 3) PROPER DEVELOPING SOLVENT :- The best
possible developing solvent is generally selected for the
separation of substances under examination. The choice of this
depends upon the simple fact that R values should be different for
different constituents present in a mixture. With Many combination
of solvent systems can be used provided they are not completely
miscible one another. The following are usual general criteria for a
good solvent system:
i. The Rf values of the sample should lie between 0.05 and 0.85
in the system.
ii. The difference between the Rf values of any two components
must be 0.05-the minimum value necessary in order to
separate any two components.
iii. The distribution ratios of the components in the solvent
system should be independent ofconcentration so as to get
circular spots
iv. The solvents should not undergo chemical reaction with any of
the components of the sample mixture.
v. The solvent should not interfere with the detection of the spots
on the developed chromatogram.

19. Solvents in their increasing polarity ;
N-hexane→Cyclohexane→carbon
tetrachloride→Benzene→Tolune→Trichloroethylene→
Diethyl ether→Chloroform→Ethylacetate→n-butanol→
n-propanol→Acetone→Ethanol→Methanol→Water.
4) PREPARATION OF SAMPLES :- It is not possible
to give any standard procedure for preparation of
samples because this problem revolves around several
factors of the given samples, especially the presence
of other systems like fats, salts, protein etc. However,
the sample volume of 10-20 μ having as many μg of
the substance is that ideal quantity to be spotted.

20. 5) SPOTTING :- For ascending technique, a strip of
Whatmann filter paper of suitable size (25 cm x 7
cm) is generally used. A horizontal line is drawn on
the filter paper by a lead pencil. This line is known
as origin line. On the origin line, cross marks (x) are
made with a pencil in such way that each cross (x)
is at least 2 cm away from each other as shown in
Fig. 32.10. By the help of a graduated micro pipette.
the test solutions are applied on cross (x) marks
and the spots are dried cautiously by a stream of
hot or cold air.

21. 6) DRYING THE CHROMATOGRAM :- THE WET
CHROMATOGRAM AFTER DEVELOPMENT ARE DRIED IN SPECIAL
DRYING CABINETS WHICH ARE BEING HEATED ELECTRICALLY WITH
TEMPERATURE CONTROLS.
7) VISUALISATION :- visualisation of the spots can be done in two
ways:
i. either by chemical means or
ii. by using physical methods
i) CHEMICAL DETECTION.:- chemical treatment can develop the
colour of colourless spots on the paper the reagents used for visualising
the spots are known as chromatogenic reagents or visualisingthe
reagents are applied in one of two ways, either by the pressure spraying
or by dipping the chromatogram. reagents in organic solvents are more
suitable for spraying than aqueous solutions then, the spots are dried.
ii) PHYSICAL METHODS.:- Some colourless spots, when held
under a UV lamp, fluoresce and rev their existence.
When the substance is coloured, the spots can be observed either by
reflected or transmitted ligh The former is more selective but the latter
more sensitive.(h) Calculation of R, Values. The distance of
chromatographed species is noted from its centre to the origin line. The
distance of solvent front from the origin line gives the Rf values.

22. 8) CALCULATION OF RF VALUES :-
The distance of chromatographed species is noted from
its centre to the origin line. The distance of solvent front
from the origin line gives the Rf values.

23. APPLICATIONS
Paper chromatography has been applied to the separation of many
organic and biochemical products. For example, it has been utilized
in the determination of indoles in whole urine and in the study of
barbiturates, antibiotics, carbonyl phosphates, hormones, and amino
acids, among others. It has also been used in the study of inorganic
metal salts and complex ions.
The locations of the migrated spots are identified readily if colored.
Otherwise, it may be necessary to detect fluorescence which is
initiated with an ultraviolet lamp or "develop" colored compounds by
spraying the separated spots with a color-forming reagent. For
example, ninhydrin is used for producing color with the amino acids.
At times it may be desirable to form colored derivatives which are
then separated and identified. chromatography has also been
employed for the analysis of mixture of sugars.

24. REFERENCES
1. https://microbenotes.com/paper-chromatography/
2. Chatwal, G.R. and Anand, S.K.J. (2011) Instrumental Methods of
Chemical Analysis. Himalaya Publishing House, Mumbai, 2.587-
2.598.
3. https://www.differencebetween.com/difference-between-ascending-
and-descending-paper-chromatography/
4. https://pharmainformationzone.blogspot.com/2020/04/descending-
chromatography.html
5. https://www.google.com/search?q=radial+chromatography&source
=lnms&tbm=isch&sa=X&ved=2ahUKEwjegpH85-
_7AhUxsFYBHdc0Aw4Q_AUoAXoECAEQAw&cshid=1670701495
402465&biw=1458&bih=606&dpr=0.94#imgrc=8Sncnbi522JPbM
6. https://www.researchgate.net/figure/1113-Ascending-Descending-
Paper-Chromatography-d-Radial-or-Circular-
Paper_fig18_339662342
7. https://www.google.com/search?q=two+dimensional+paper+chrom
atography&source=lnms&tbm=isch&sa=X&ved=2ahUKEwjvzqP16
O_7AhXYrVYBHVDXAegQ_AUoAXoECAEQAw&biw=1458&bih=6
06&dpr=0.94#imgrc=XLpIbNm9wKEMyM