Paper chromatography is a physical technique used to separate and analyze components in mixtures. It works by partitioning components between a stationary phase (paper) and mobile phase (solvent). Key steps include spotting the sample, developing the chromatogram by allowing the mobile phase to travel up the paper, drying the paper, and analyzing the separated components. Paper chromatography is useful for separating and identifying various substances like amino acids, drugs, and organic compounds. It has applications in fields like pharmacy, biochemistry, and research due to being inexpensive and easy to perform.

1. Assignment of analytical chemistry
Paper Chromatography
Paper chromatography is a physical technique used for quantitative and
qualitative analysis of components by separating them from complexes and
mixtures.
 Instructor :
Miss SeharFatima
 Submitted by:
Tehreem Abbas : BS BT 13-F10
Maryam Gillani : BS BT 13-F14
Aiman Mahmud : BS BT 13-F18
SaniaShokat : BS BT 13-F25
Momal Babar : BS BT 13-F29
PernianDhillon : BS BT 13-F40
Rizwan Abbas : BS BT 13-F43
ToobaShahzad : BS BT 13-F45
2015
Submitted to:Miss Sehar Fatima
Bzu Lahore campus
3/18/2015
Department of Biotechnology

2. PAPER CHROMATOGRAPHY
Table of contents
S.no Title Sub titles Page
A
Introduction.  Chromatography
 History 01
B
Paper chromatography  Introduction
 principles
02
C
Experimental steps  types
 apparatuses and chemicals
04
D
Procedure
 steps
 calculation 5
E
Development
 techniques of development
 analysis
6
F
Applications
 uses of paper chromatography
9

3. Chromatography
Chromatography is a non-destructive physical technique for separating and identifying the
components of a mixture through two phases.
Or
Chromatography may be defined as a method of separating a mixture of components into
individual components through equilibrium distribution between two phases a stationary
phase and a mobile phase.
History
 In 1850-1900Runge, Schoenbein, and Gospel Schroeder Studied capillary analysis
on paper for separation of different components of mixture for the first time.
 Chromatography is relatively a new technique which was first invented by M. Tswett,
a botanist in 1906 in Warsaw. In that year, he was successful in doing the separation
of chlorophyll, xanthophylls and several other colored substances by percolating
vegetable extracts through a column of calcium carbonate acted as an adsorbent and
the different substances got adsorbed to different extent and this gives rise to colored
bands at different positions, ion the column.
 Tswett termed this system of colored bands as the chromatogram and the method as
chromatography after the Greek words chroma and graphs meaning “colour” and
“writing” respectively.
 In 1939 Brown first time showed paper chromatography and in 1944Consden,
Gordon, and Martin First described paper partition chromatography.
PAPER CHROMATOGRAPHY
It is defined as a technique in which the analysis of an unknown substance is carried out
mainly by the flow of solvent (mobile phase) on specially designed fitter paper (stationary
phase).
Paper chromatography is a technique used for the separation & identification of relatively
small chemical substances by a moving solvent on sheets or strips of filter paper. As the
solvent moves up the paper, different molecules move at different rates with the smallest
molecules moving the fastest. The separation is effected by differential migration of the
mixture of substances, which takes place due to difference in partition coefficients
Paper chromatography is one of the types of chromatography procedures which run on a
piece of specialized paper. It is a planar chromatography system wherein a cellulose filter
paper acts as a stationary phase on which separation of compounds occurs.
Basic principles
All chromatographic methods require one static part (the stationary phase) and one moving
part (the mobile phase). The techniques rely on one of the f phenomena: adsorption; partition;
ion exchange; or molecular exclusion.
Partition chromatography
The principle involved is partition chromatography where in the substances are distributed or
partitioned between to liquid phases. One phase is the water which is held in pores of filter

4. paper used and other phase is that of mobile phase which moves over the paper. The
compounds in the mixture get separated due to differences in their affinity towards water (in
stationary phase) and mobile phase solvents during the movement of mobile phase under the
capillary action of pores in the paper.
Chromatography using a non-volatile liquid stationary phase held on an inert solid surface is
known as partition chromatography. The components distribute themselves between the two
phases according to their relative solubility. Paper chromatography and gas liquid
chromatography are examples. The more soluble or volatile the component, the faster it will
move
Adsorption chromatography
The principle can also be adsorption chromatography between solid and liquid phases, where
in the stationary phase is the solid surface of paper and the liquid phase is of mobile phase.
But most of the applications of paper chromatography work on the principle of partition
chromatography i.e. partitioned between to liquid phases.
Chromatography which uses a solid stationary phase and a mobile liquid or gas phase is
known as adsorption chromatography. Some components of the mixture are attracted to the
solid surface and the other components which are less strongly bonded travel faster with the
mobile phase. Thin-layer chromatography is an example. As the stationary phase is generally
a polar solid, the more polar solutes are more readily adsorbed than the less polar solutes.
Apparatus and Chemicals NeedGenerally.
 Beaker or small container (for water)
 Pencil tip (or sharp object such as a push pin)
 Chromatography centrifuge device Pipettes, Beral-type
 Filter paper, 12.5-cm diameter
 Plastic rotating disk (with small hole in center)
 Markers, water-soluble (or felt-tip pens)
 Water, tap
Types of paper chromatography basically there are two main types of paper
chromatography on basis of the solvent and phases apply. Normal phase and Reverse phase
which are applicable on all type of principal techniques.
 Normal phase. In this type Stationary phase and the solute should be polar and that
mobile phase be non polar,
Usually applied in partition and ion exchange method
 Reverse phase. In this type reverse of normal phase is applied
Generally reverse phase applied in absorption principal with modification of paper

5. GeneralProcedure
 Obtain a piece of filter paper. Determine the center of the piece of filter paper.
 Set the piece of filter paper onto the plastic disk and mark the center hole.
 Use a sharp pencil tip or push pin to puncture a small hole into the center of the piece
of filter paper.
 Using a black (or dark-colored) water-soluble marker, draw four to six small dots in a
circular or random pattern around the center hole in the filter paper.
 Place the plastic rotating disk (with a center hole) onto the exposed motor axle of the
chromatography centrifuge.
 Set the filter paper on the disk. The axle should penetrate the center hole of the filter
paper to secure the paper to the rotating disk.
 Start the motor running by completing the circuit on the centrifuge using the attached
alligator clips.
 Watch the disk rotate around in a circle. Using a Beral-type pipette, add a few drops
of water on the center of the rotating piece of filter paper. Add additional (yet
minimal) drops of water if necessary.
 Watch carefully and make observations as the filter paper rotates on the centrifuge.
The radial chromatogram is done when the water line is near the edge of the filter
paper.
 Disconnect the clips. Lay the disks flat to dry.
Separation parameters
The positions of separation of spots on the Chromatogram are indicated by terms such as RF<
retention factor>
RF the R is related to the migrations of solute front from solvent front.
RF = distance travelled by the solute from the origin line distance travelled by the solvent
from the origin line. It follows capillary action depending on type of paper, duration and direction of
development, amount of liquid in the reservoir, humidity etc.
I.e. quality of paper
 Nature of mixture.
 Temperature and,
 The size of the vessel in which the operation is carried.
RF= distance moved by solute
Distance moved by solvent
chromatography
tank
lid
chromatography
paper
origin
solvent
solvent
front
spots
x
y
Rf = x/y

6. Experimental methods .
The experimental method involves following steps.
1. Selection of suitable type of development: This depends on complexity of the
mixture, solvent, paper etc. But in general ascending type or radial type of
chromatography are used as they are easy to perform, handle, less time consuming
and also give chromatogram faster.
2. Selection of suitable filter paper: Filter paper is selected based on pore size, quality
of the sample to be separated and also mode of development.
3. Preparation of sample: Preparation of sample involves dissolution of sample in
suitable solvent used in making mobile phase. The solvent used should be inert with
the sample under analysis.
4. Spotting of sample on the paper. Samples are to be spotted at proper position on the
paper using preferably a capillary tube.
5. Development of chromatogram: Sample spotted paper is subjected to development
by immersing it in the mobile phase. The mobile phase moves over the sample on the
paper under the capillary action of paper.
6. Drying of the paper and detection of the compounds. Once the development of
chromatogram is held the paper is held carefully at the borders so as to avoid touching
the sample spots and dried using an air drier. Sometimes the detecting solution is
sprayed in the developed paper and dried to identify the sample chromatogram.
Techniques of development Based on the way the development of chromatogram on
the paper is done in the procedures, we have broadly 7 types of chromatography.
1. Ascending chromatography
2. Descending chromatography:
3. Radial mode:
4. Horizontal
5. Spiral
6. Two dimensional chromatography and
7. Wedges strip
A. Ascending chromatography
As the name indicates, the chromatogram ascends. Here the development of paper occurs due
the solvent movement or travel in upward direction on the paper. When the development of
the paper is done by allowing the solvent to travel up the paper it is known ascending
technique. The solvent reservoir is at the bottom of beaker. The paper tip with sample spots
just dips into the solvent at bottom such that spots remain well above the solvent.

7. B. Descending development. Here the development of paper occurs due to solvent travel
downwards on the paper. The solvent reservoir is at the top. The movement of solvent
is assisted by gravity besides capillary action. The advantage of descending technique
is that the development can be continued indefinitely even though the solvent runs off
at the other end of paper.
C. Radial technique Here the solvent travels from center (mid point) towards
periphery of Circular chromatography paper. The entire system is kept in a
covered Petri dish for development of chromatogram. The wick at the center of
paper dips into mobile phase in a Petri dish by which the solvent drains on to
the paper and moves the sample radially to form the sample spots of different
compounds as concentric rings.
D. Two dimensional modes. Two-dimensional paper chromatography was first
described by Consden, Gordon and Martin for the separation of 20 amino acids. It is
used for large numbers of substances separation 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.
Mixtureof
amino acids
on origin line
Paper driedand
rotatedclockwise
through90 o
Solvent
front
First solvent
Secondsolvent
Two- way chromatography providesbetter separationof substancesthat behave
in a similar fashion in thefirst solvent.
A secondrun in a different solvent resolvestwo very closespotsmoreclearly
Solvent
front

8. E. Ascending – Descending/Spiral mode It is a hybrid of two techniques. The
upper part of ascending chromatography can be folded over a glass rod allowing the
descending development to change over into the descending after crossing the glass
rod. The chromatogram is repeatedly developed in the same direction and thus the
complete resolution of two or more substances which have Rf values close together can
be obtained. As the mobile phase one can use either the same solvent system or
different solvent systems
F. Horizontal technique, This technique chromatography closely resemble to those of
column and paper chromatography. The components are separate horizontally as
solvents movement is.
G. Wedge strip method In this method of development a strip of paper is used vertically. It
works on basis of adsorption principle. As the paper absorbs the solvent, the soluble
components of the mixture are carried away by the moving solvent which acting as mobile
phase.
Analysis In the last step the analysis is carried out by two methods as there are two type of
analysis may be necessary
Quantitative and Qualitative

9. A. Quantitative analysis
It is measure of the amount of the components separated on chromatogram which can
be determined by following methods.
 Densitometry
 Radioactive techniques
 Conductometricmethod
 Polarographic mode (UV absorbance)
Qualitative analysis
The quality or nature of the separated components on chromatogram can be judge by
different methods according to the character of those constituents.
 Colored components are visually detected and judgments is easy
 Uncolored constituents can be detected by physical and chemical method which is
non destructive and destructive modes respectively.
 Physical / non destructive modes E.g. UV detection
 Chemical method is destructive type by using chemicals as locating reagents
 Locating reagents are of two types (specific and non specific)
NinHydrin is specific locating reagent which form colored complexes with amino
acids
2, 4Di nitro phenol hydrazine identifies carbonyl compounds
Ammonia and Sulphuric acids are non specific reagents which identifies the major
organic compounds.
Uses and applications of paper chromatography:
Some applications of paper chromatography are
 Paper chromatography is specially used for separation of mixture having polar and
non-polar compounds.
 For separation of amino acids.
 It is used to determine organic compounds, biochemical in urine etc.
 In pharmacy sector for determination of hormones, drugs.
 Sometimes used for evaluation of inorganic compounds like salts and complexes.
 Easily available and cheapest technique applicable for research.

10. References
The data on topic paper chromatography are collected from following sources
 www.wikipedia.com/paperchromatography
 www.yahooanswers.com
 A text book of analytical chemistry by S. Chand publisher/chapter1/Chromatography
 A lab manual of chemistry booklet by C.G.Rasool
 www.GoogleScholaralerts.com/applicationsofchromatography2004.atr13
 www.ncbi.com/analysis/applicationsofchromatography
The End