This document discusses paper chromatography techniques. It begins with definitions of chromatography and a brief history. It then explains the principle of paper chromatography, which involves the separation of compounds through their partitioning between a stationary phase (filter paper) and mobile phase solvent. The document outlines the basic experimental procedure for paper chromatography and describes various development techniques, including ascending, descending, radial, and two-dimensional modes.

1. BS. Biotechnology
Instructor
Miss Sehar Fatima
Submitted By:
Rizwan Abbas (BsBt-13-f-043)
BAHAUDDIN ZAKARIYA UNIVERSITY LAHORE CAMPUS
PAPER CHROMATOGRAPHY
Assignment of Analytical Chemistry and Instrumentation
ABSTRACT;
Paper chromatography is a physical technique used for quantitative and qualitative analysis of
components by separating them from complexes and mixtures. A short assignment on techniques
of paper chromatography presented to Miss Sehar Fatima on due date 04 –march.
The data collected from source given as references within the table of contents

2. 1
PAPER CHROMATOGRAPHY
Content and references
Tittle Reference Page
A Introduction.
.
A text book of Chemistry
By PTB 01
B Explanation Analytical chemistry by F.W.Fifield and
D. Keeley 01
C Types A text book of Chemistry
By PTB 02
D Experimental steps www.wikihow.com /chromatography?
03
E Procedure www.wikipedia .com articles/chem/.pk 04
F Techniques of development Analytical chemistry by F.W.Fifield and
D. Keeley
www.science
alert.com/articles/instrumentations
06

3. 2
PAPER CHROMATOGRAPHY
Definition:
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.
History:
In 1850-1900 Runge, Schoenbein, and Goeppel sroeder 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, xanthophyii and several other colored substances by percolating vegetable
extracts through a column of calcium carbonate.
In 1939 Brown first time showed paper chromatography and in 1944Consden, Gordon,
and Martin First described paper partition chromatography
Explanation:
Paper chromatography is one of the types of chromatography procedures which runs on
a piece of specialized paper. It is a planar chromatography systems wherein a cellulose
filter paper acts as a stationary phase on which separation of compounds occurs.
Principle of paper 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 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.
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.
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 pharma 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.

4. 3
Types of paper chromatography
There are two main types of chromatography: partition and adsorption chromatography.
Partition chromatography
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
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 Need Generally.
Beaker or small container (for water)
Pencil tip (or sharp object such as a push pin)
Chromatography centrifuge device Pipets, 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
Paper chromatography experiment method.
The experimental method involves following steps.
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.
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.
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.
Spotting of sample on the paper.
Samples are to be spotted at proper position on the paper using preferably a capillary
tube.
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.

5. 4
Drying of the paper and detection of the compounds.
Once the development of chromatogram is over. 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.
General Procedure
1. 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.
2. 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.
3. 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.
4. 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 pipet, 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.
5. 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.
6. Disconnect the clips. Lay the disks flat to dry.
Separation parameters
1. The positions of separation of spots on the Chromatogram are indicated by
terms such as RF < retention factor>
2. RF The R is related to the migrations of solute front from solvent front.
3. RF = distance travelled by the solute from the origin line distance travelled by the
solvent from the origin line.
4. It follows capillary action.
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. Ascending- descending mode. Spiral
4. Radial mode:
5. Two dimensional chromatography
6. Wedges strip
7. Horizontal/linear

6. 5
A. Ascending Development
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.
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.

7. 6
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 petridish for development
of chromatogram.
The wick at the center of paper dips into mobile phase in a petridish 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 mode.
Where separations were not achieved in a single development, it was often
possible to achieve the desired result using a second solvent system of different
composition and development in a second dimension at 903 to the original
direction of chromatography. Two-dimensional paper chromatography was first
described by Consden, Gordon and Martin for the separation of 20 amino acids,
but was subsequently widely employed.
.
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.

8. 7
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
THE END