PROF.

NAVEED ASLAM DOGAR , Assistant Professor of Chemistry, GCS, LAHORE (03224976417)

M.SC( Chemistry), M.phil (Chemistry), M.Phil (Environmental Sciences), CSS(2010), PhD (Scholar) GCU, Lahore.

PAPER CHROMATOGRAPHY FOR M.SC & B.S(Hons)

PAPER CHROMATOGRAPHY
BASIC PRINCIPLE
Paper chromatography is an important and useful class of partition chromatography. In
this technique stationary phase is made up of water molecules bonded to cellulose
network of the paper the mobile phase can be a solvent or a mixture of different solvents
which are miscible into each other. The mobile phase can also be called as developing
solvent. Separation of the components of mixture takes place by the partition of these
components in two different phases. In paper chromatography mobile travels by capillary
action through the paper. Depending upon the way of solvent travels on the paper, there
are three types of chromatography.
• Ascending paper chromatography
• Descending paper chromatography
• Radial paper chromatography

In ascending paper chromatography mobile move up on paper strip. In descending paper

chromatography mobile phase moves down the paper the paper strip and in radial paper
chromatography mobile phase moves horizontally along the circle.

STEPS FOR PAPER CHROMATOGRAPHY

Technique of paper chromatography is very simple. The various steps involved in paper
chromatography are as follows
▪ Choice of filter paper
▪ Preparation of solution or sample
▪ Application of sample on paper
▪ Choice of solvent
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PROF. NAVEED ASLAM DOGAR , Assistant Professor of Chemistry, GCS, LAHORE (03224976417)
M.SC( Chemistry), M.phil (Chemistry), M.Phil (Environmental Sciences), CSS(2010), PhD (Scholar) GCU, Lahore.

▪ Development of chromatogram
▪ Drying the paper
▪ Location of substance on the paper
▪ Quantitative estimation

CHOICE OF FILTER PAPER

Generally what’s man filter paper is extensively used in paper chromatography. What’s
man filter paper is composed of following compounds:
• Alpha-cellulose (98.9% )
• Beta-cellulose (0.3% to 1.0%)
• Pentosans ( 0.4% to 0.8% )
• Ash ( 0.07% to 0.01% )
• Ether soluble substance ( 0.015% to 0.1% )

A suitable filter paper should have following properties:

• Excellent rate of movement of water
• Negligible difference of the movement of spot
• Maximum degree and clarity of separation

The most common shape of paper is rectangular. Although square shaped filter paper can
also be used. Usually the length of the filter paper should be between 15 to 30 cm and
width is one to several centimeters.
PREPARATION OF THE SOLUTION
▪ Choice of a proper solvent for making sample solution is very important step in
paper chromatography.
▪ The choice of a solvent entirely depends upon the nature of the substance to be
separated. In general, polar solutes are dissolved in polar solvents and non-polar
solutes are dissolved in non-polar solvents.
▪ Pure solutions can also be applied directly on the filter paper but solids are always
dissolved in a small quantity of suitable solvent.
▪ As biological tissues are treated with suitable solvents and their extracts are
obtained. Since large amount of proteins and salts are present in biological
extracts and they may interfere with the partition process. It is therefore, necessary
to remove unwanted (molecules) materials from the extract.
▪ Protein can be removed by ion exchange resin or electrolyte method. Concentrated
solutions are usually applied on the filter paper to avoid diffusion through the
paper.

APPLICATION OF SAMPLE ON THE FILTER PAPER

▪ Application of the sample on the filter paper is also an important step. A platinum
lope is usually used for qualitative purpose. It is also preferred for the practical
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PROF. NAVEED ASLAM DOGAR , Assistant Professor of Chemistry, GCS, LAHORE (03224976417)
M.SC( Chemistry), M.phil (Chemistry), M.Phil (Environmental Sciences), CSS(2010), PhD (Scholar) GCU, Lahore.

purpose because it can be used again and again and by careful washing and
heating strongly in a flame after each application.
▪ Micro-pipettes and micro-syringes are available in many sizes and can be used for
qualitative as well as quantitative purpose.
▪ In some case sample can also be applied with the help of capillary tubes.

CHOICE OF A SOLVENT
The nature of the solvent plays an important role in the development of paper
chromatogram. The solvent should be free from impurities and dried before use. Polar
solvents such as water is adsorbed by the paper and held stationary whereas less polar
solvents such as ethanol, acetone, glycol, acids and amines flow through easily. Although
pure solvents is used but a mixture of solvents preferred. The solvent should have
following properties:
a) The solvent should not react chemically with any of the components of the mixture.
b) The composition of the solvent mixture should not change with time.
c) The solvent should not interfere with the detection of spots.
d) The minimum difference between the Rf value of any two components should be from
0.05 to 0.1.

The various types of solvents used for the separation of different components are as follows
Separators Solvents
Proportions

Amino acids n-butanol /acetic acid / water 4:1:5

n-butanol /pyridine / water 1:1:1
Sugars Ethyl acetate /pyridine /water 2:1:2
Ethyl acetate /acetic acid /water 14 : 3 : 3
Fatty acids n-Butanol /1.5M Ammonia saturated solution
Co, Ni, Mn, Acetone /conc. Hydrochloric acid /water 85 : 8 : 5
Cu, Fe (chlorides)

DEVELOPMENT OF CHROMATOGRAM

After the location of the components of the mixture, a pattern which we get is called
chromatogram. The development of chromatogram depends upon the accuracy of the
previous steps. After development of chromatogram, it is dried by hanging the paper in
an open environment or by drying it with the help of dryer. Before drying the paper , we
have to mark the solvent front so that we may easily calculate the Rf values of the
different components of the samples.

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PROF. NAVEED ASLAM DOGAR , Assistant Professor of Chemistry, GCS, LAHORE (03224976417)
M.SC( Chemistry), M.phil (Chemistry), M.Phil (Environmental Sciences), CSS(2010), PhD (Scholar) GCU, Lahore.

LOCATING THE SUBSTANCE

After the development of chromatogram the solvent front is marked and solvent is dried.
Spots of the separated compounds are detected in different ways. If the samples are
visible, their Rf values are measured directly but when the spots colorless we have to use
some methods to locate the spots.
There are three methods locating the spots:
➢ Physical method
➢ Chemical method
➢ Radioactive method

Physical method
Physical are more beneficial than chemical methods because of the fact that the
substance on the paper can be converted into other substance. If the substances are not
visible in ordinary light then U.V. lamps are used to locate the spots.
Chemical method
Chemical methods can be used where physicals methods cannot be used. Reagents that
form compounds in the presence of certain functional groups are available. For example,
the locating agent for amino acids is Ninhydrin which can make purple or blue complexes
with amino acids. The locating agents are the reagents which give a color reaction
with sample components. The locating agents are applied by spraying the solutions on
the paper or dipping the paper in reagent solution.
Locating agents for different
substances
Metal locating agents
• Rubeanic acid
• Di-phenyl carbazide
• Ammonium sulphide
Amino acid locating agents
• Ninhydrin
• Isatin
• Sakaguchi
Sugar locating agents
• Silver nitrate
• Aniline
• Anisaldehyde
Radioactive method
The location of substance can also be carried out by using radioactive substance.
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PROF. NAVEED ASLAM DOGAR , Assistant Professor of Chemistry, GCS, LAHORE (03224976417)
M.SC( Chemistry), M.phil (Chemistry), M.Phil (Environmental Sciences), CSS(2010), PhD (Scholar) GCU, Lahore.

RETARDATION FACTOR / QUANTITATIVE ESTIMATION

The rate at which a solute has moved is determined by its retardation factor. The
retardation factor can be defined as the ratio of distance travelled by the component
from base line to the distance travelled by the solvent from base line.
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡
Retardation factor =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡
▪ The value of Rf is always between the range of 0 to 1. The maximum value of Rf is 1
which is observed for a salute migrating at the same velocity as the mobile phase.
▪ The minimum value of Rf is zero which is observed when solute spends all the time in
the stationary phase and remains in its original position on the filter paper.
▪ Rf values are useful if they are between two extremes values. For good resolution
reasonable Rf value must be in the range of 0.4 to 0.8. The relationship between Rf value
and k value is
1
Retardation factor =
𝑘+1
Here k is distribution coefficient and it is given by
𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒
k=
𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑖𝑛 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒
✓ If the value of k is large then the concentration of component in mobile phase is large.
✓ If the value of k is small then the concentration of component in stationary phase will
large.
✓ If the value of k is average then the concentration of component will be equal in
stationary phase and mobile phase.

It has been observed that Rf values are influenced by the impurities in paper, solvent,
temperature and the saturation of environment.

There are four factors which effect the Rf values.

a) Nature of the paper
b) Solvent system
c) Nature of sample
d) Development process

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PROF. NAVEED ASLAM DOGAR , Assistant Professor of Chemistry, GCS, LAHORE (03224976417)
M.SC( Chemistry), M.phil (Chemistry), M.Phil (Environmental Sciences), CSS(2010), PhD (Scholar) GCU, Lahore.

(a) Nature of the paper

▪ The type of paper can affect the Rf value. More dense paper will have a large
volume of the stationary liquid phase. In lose paper, more of the mobile liquid will
be able to penetrate the fiber structure so Rf values are lower in more dense paper
and higher in less dense paper.
▪ In addition to cellulose filter paper usually have small mineral contents. The
presence of minerals can affect the movement of solute through the paper by
chemical reaction. The mineral content is about
▪ The presence of traces of Ca or Mg can disturb the separation of phosphate by
forming insoluble phosphate. Similarly copper and iron can form complex with
amino acid and can hinder the separation process. The storage of filter paper can
also effects its properties not only humidity but dust, dirt and heat can alter the
properties of paper.

(b) Solvent system

▪ The chemical and physical properties of solvent can affect the Rf values. If a pure
liquid is used the problems are less complicated. In paper chromatography, the
developing solvent is not a pure solvent but a mixture of liquids. The use of these
mixed solvents can create new problems. For example, each substance moves
along the paper at its rate by capillary action.
▪ Thus in a mixture of solvents each of the component move from its starting line at
its flow rate as a result the composition of the solvent changes continuously.
Another problem is different vapor pressures of components unless the developing
chamber is saturated the rate of evaporation of component of solvent will remain
different.
▪ Sometimes when chemicals are mixed together, the chemical reaction takes place.
For example, when n-butanol, acetic acid and water are mixed together as a
solvent they react with each other to form ester.

(c) Nature of sample and Development Process

▪ Paper chromatography is used to identify the components in a variety of samples.
Many of these samples are complex mixtures of biological reagents, mixture of
reaction product and commercial product.
▪ The fluid of unknown samples may contain many substances which can alter Rf
value. The fluid which is more viscous can reduce the capillary action rate. This will
lead to the chemical reaction of sample components. For example the amino acids
in alcoholic extracts of plants can react with solvents like ethanol to form amino
acid esters. These esters have different Rf value as compared to free amino acids
value.
▪ The application of sample can also change Rf value. It should not be expected to
find the same Rf values if different procedures are followed. For example, in

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PROF. NAVEED ASLAM DOGAR , Assistant Professor of Chemistry, GCS, LAHORE (03224976417)
M.SC( Chemistry), M.phil (Chemistry), M.Phil (Environmental Sciences), CSS(2010), PhD (Scholar) GCU, Lahore.

ascending, descending, radial, horizontal and spiral development of the same

sample in same solvent can have slightly different Rf values.

APPLICATIONS OF PAPER CHROMATOGRAPHY

Following are the useful applications of paper chromatography

1. It is used for the identification of trace metals in ores.
2. It is also used in the separation and identification of cobalt, nickel and zinc.
3. It can be used to check the purity of pharmaceutical and fermentation products.
4. It can be used to separate the mixture of pesticides ( dichlorodiphenyltrichloroethane,
benzene hexa-chloride, Heptachlor, Endrin and Aldrin).
5. It can also be used for the specification of different anions of sulphur(e-g S2-1,SO32-,SO42
and S2O32- ).
6. It can also be used for detection of adultrance and contaminants in food and drinks.
7. It can be used for the separation of amino acids.