Paper Chromatography

• Martyn, Consden, Gorden and Synge have developed

this technique in 1941.
• Advantages of paper chromatography:
➢ The equipment is very simple and easily available
➢ It has high efficiency of separation
➢ Separation can be done on micro or semi micro scale
➢ Closely related homologous, isotopes, isomers can be
separated
Theoretical Principle:
• Separation by paper chromatography is largely by partition
coefficient phenomenon. The separation is accomplished by
successive equilibrations of sample between two phases.
The stationary phase is made up of the solvent held by the
paper and mobile phase is the irrigating eluent.
• Rf = Distance traveled by the solute / Distance traveled by
the solvent front
Factors affect the Rf value are
➢ The solvent system and its composition
➢ Temperature
➢ The quality of the paper
➢ Distance and quality of solvents used
➢ The method of development
Types of paper chromatography:
• Paper partition chromatography: The paper is utilized as
a support with one solvent as mobile phase and other as a
stationary phase. The migration of substance is due to
differences in partition coefficient

• Paper adsorption chromatography: In some cases the

paper is coated with adsorbents like silica gel or alumina.
However this technique is not much used.
Operational Technique:
1. Choice of Filter paper: Whatman filter papers are used
extensively. The filter paper contains 98-99% of alpha
cellulose. The mineral content may vary from 0.07-
0.01%. Besides this beta cellulose, ether soluble matter,
ammonia and lipophilic substances (waxes, fats) are also
present. They are supplied in packs of 100 or 500 sheets
46 x 57 cm or 58 x 68 cm size. The rectangular or square
papers are cut for separation. Whatman paper number
31 is four times faster than Whatman paper number 1.
• Modified Filter papers: For efficient separation
of certain substances, specially treated or
modified filter papers are used i. e. Buffered or
treated papers like Whatman – phosphate,
Whatman – citrate or paper treated with silicic
acid, alumina etc.
• Preparation of paper: Once the type of paper
is decided, it is cut in desired size and shape.
Generally, rectangle and square shapes are
used. While storing paper should be kept
away from any fume (especially ammonia) and
should not be kept in area where large
changes in humidity occurs.
• Preparation of sample: The mixture to be
separated is applied to paper as solution. A
weighed amount of mixture is dissolved in
volatile solvent and a minimum volume of
concentrated solution is applied on the paper.
Sample volumes of 10 – 20 microlitres
containing as many micrograms of substances
are spotted.
• Application of sample: The straight line is marked on
the paper with an ordinary pencil some 5 cm form the
bottom edge. On the straight line marks are made
about 2 cm apart from each other. Micro pipette, glass
capillary or micro syringe is used for application of
sample. The spot diameter should not exceed 5 mm.
The quantity of sample applied on paper is important
than the volume. When solution is very dilute it can be
concentrated on paper by applying series of drops to
the same spot, allowing each drop to evaporate before
applying next. Drying of spotted chromatogram should
be carried out carefully in air.
• Solvents: The selection of proper solvent
depends on nature of substances to be
separated. The solvent should be cheap and
very pure. The solvents are given in increasing
order of polarity as n –hexane, cyclohexane,
CCl4, Benzene, Toluene, Diethyl ether,
Chloroform, Ethyl acetate, n-butanol, n-
propanol, Acetone, ethanol, methanol, water.
• Chromatographic Tank or Chambers: The
chromatographic tanks are made from various
materials like glass, plastic or stainless steel. Glass
tanks are preferable and most commonly used.
They are available in various dimensional sizes
depending upon the length and breadth of paper
and type of development. The tanks have a lid
with hole for introducing the solvent through it.
Saturation of chamber with solvent is carried out
by using filter paper dipping in solvent system.
• Development of Chromatogram: For carrying out proper
development following points are considered
i) Sufficient amount of solvent should be present in chamber
ii) During development, paper should be freely suspended.
iii) Large temperature changes and exposure to light should be
avoided.
Iv) The chamber atmosphere should be saturated with solvent
vapor.
v) The paper is dipped in solvent in such a manner that the
spots will not dip completely into the solvent.
vi) The solvent will run over the paper by capillary action.
• Development techniques:
• Descending Chromatography: The apparatus consists of a
well sealed glass tank of suitable shape and size provided with
a trough for the mobile phase in the upper portion. The paper
with the sample spotted is inserted in trough containing
mobile phase. The movement of mobile phase is from top to
bottom.
• Ascending Chromatography: The mobile
phase is placed in chamber at bottom. The
samples are applied a few centimeters from
the bottom edge of paper. The movement of
mobile phase is from bottom to top.
• Ascending – Descending Chromatography: A hybrid of the
above two techniques is called ascending-descending
chromatography. The upper part of the ascending
chromatogram can be folded over a glass to change over into
the descending after crossing the glass rod.
• Radial or Disk Chromatography: In this case a circular piece of
paper is taken has a wick cut parallel to the radius from the
edge to the centre. The sample is deposited at the centre of
the paper and at the upper end of the wick. The paper is then
laid on the edge of a circular disk with the wick dipping into
the solvent at the bottom of the dish. The liquid ascends by
the wick and flows radically through the paper. It carries the
compounds with it. But this requires more time and this is
used rarely.
• Multiple Chromatography: In this the irrigation is repeated in the same
direction as in the first run or in direction perpendicular to the first.
• Two-Dimensional Chromatography: In this method the paper is cut
square or rectangle. The sample is applied to one of the corners. Using
solvent system, first development is carried out as per ascending method.
The paper is taken out and dried. The second development is performed
at right angle to the direction of the first using other solvent system.
• Drying of Chromatogram: The wet chromatogram was
dried by cold or hot air depending upon the volatility of
solvent. A simple hair dryer is a convenient device to dry
chromatograms.
 DETECTING / VISUALISING AGENTS
If the substance are colored they are visually
detected easily.
But for colorless substance, Physical and chemical
methods are used to detect the spot.
(a) Non specific methods ( Physical methods)
E.g. iodine chamber method,
UV chamber for fluorescent compounds – at 254 or
at 365nm.

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(b) Specific methods (Chemical methods) or Spraying
method - examples,

• Ferric chloride • Phenolic comp. &

tannins
• Ninhydrin in acetone • Amino acids
• Dragendroff’s reagents • Alkaloids
• 3,5 dinitro benzoic acid • Cardiac glycosides

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• Evaluation of chromatogram:
• Qualitative analysis: The simple measurement of Rf
value of sample is done by compared with the reference
substance or the standard values from literature.
• Quantitative analysis: The quantitative method can be
done by either estimation of the amount of substances
in the spot on the paper or by removal of the
substances from the paper and analysis of the separate
fractions by conventional quantitative techniques. The
Methods can be divided into two main groups.
➢ Evaluation of substance on the paper directly
➢ Removal of substance from the paper (Elution method)
• Evaluation of substance on paper
• Visual Comparison of spots: In this number of
chromatograms are run on same sheet with the
reference solution containing known amount of
substance. The spot is compared for its size and intensity
of color.
• Measurement of Area of spot: There is linear
relationship between the amount of substance and area
of the spot. The area can be measured by planimeter or
a graph paper.
• Radiotracer analysis: The radioactive element is used to
locate the quantity of material on the chromatogram.
The compound is identified by subjecting to neutron
bombardment. The location is measured by passing the
paper either in gas flow counting chamber or a thin
window Geiger Muller tube.
• Removal of substance from the paper: The
substance is removed by elution from paper.
The procedure is to cut out the part of filter
paper and soak it in minimum amount of
solvent. Extraction can be made by shaking or
warming. The elute so obtained diluted or
concentrated and then analyzed.