paper chromatography

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paper chromatography
At first what is chromatography ?
Chromatography is a technique by which natural compounds
of different chemical structure are separated by differential
migration between two different phases .
Paper chromatography :
 Paper chromatography (PC) is a type of a planar whereby
chromatography procedures are run on a specialized paper.
 PC is considered to be the simplest and most widely used of
the chromatographic techniques because of its applicability
to isolation, identification and quantitative determination
of organic and inorganic compounds.
 It was first introduced by German scientist Christian
Friedrich Schonbein (1865).
Types of Paper chromatography :
1.Paper Adsorption Chromatography : Paper impregnated
with silica or alumina acts as adsorbent (stationary phase)
and solvent as mobile phase.
2.Paper Partition Chromatography : Moisture / Water
present in the pores of cellulose fibers present in filter
paper acts as stationary phase & another mobile phase is
used as solvent In general paper chromatography mostly
refers to paper partition chromatography.

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Principle of Paper chromatography :
The principle of separation is mainly partition rather than
adsorption. Substances are distributed between a stationary
phase and mobile phase. Cellulose layers in filter paper contain
moisture which acts as stationary phase. Organic
solvents/buffers are used as mobile phase. The developing
solution travels up the stationary phase carrying the sample
with it. Components of the sample will separate readily
according to how strongly they adsorb onto the stationary
phase versus how readily they dissolve in the mobile phase.
Paper chromatography works in few steps:
Step 1: A horizontal line is drawn near one end (about 1.5 cm from the
bottom edge) of the paper. In figure below 6 is the horizontal line.
Step 2: The sample needs to be separated is placed as a small drop or
line on to the paper using capillary tube. Labelling the drop by a pencil
with an alphabet or number help to identify the compound later. In
figure above 3 and 4 are the drops labelled. The drops are then soaked
on the paper and dried.
Step 3: The paper is then placed into a sealed container with a swallow
layer of suitable solvent. The solvent level must be lower than the
pencil line or drop on it. The container need to be covered to stop the
solvent to evaporate.
Step 4: The solvent rises up the paper chromatography taking each
component of the sample with it. The components travel with the
solvent depends on three things:
 The polarity of the sample molecule. The non polar components
travel faster than the polar component.

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 The attraction between the sample molecule and the solvent or
solvent mixture.
 The attraction between the sample and the silica.
Step 5: When the solvent rises near the end of the paper then the
paper should be taken out from sealed container and air dried. The
paper with separated bands of components are then observed under
UV-light.
Then there is a several steps are require in paper
chromatograic technique :
1-Choice of paper :
whatman no1 filter paper are used for analytical purposs
whatman no2 filter paper are used for preparative pc
2- Choice of solvent :
Different combinations of organic and inorganic solvents
may be used depending on the analyt .
Ex :Butanol: Acetic acid: Water (12:3:5) is suitable solvent for
separating amino-acids.
3-Sample purification :
Impure extracts contain resinous and colouring matters
which interfere with the separation so, the extract must be
purified either by column or by liquid – liquid extraction.
4-Development :
After application of the sample to paper like what we do in
page 2 then placed the paper in the Jar wiat for
equilibration, after equilibration the mobile phase is
introuduced and development is allowed .

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Modes of development :
1-Assending method :
 Paper is stood vertically and immersed in the
mobile phase. The level of the spots should be
above the liquid by 1-2 cm.
 The paper may be suspended from a hock
attached to the stopper or bent into a cylindrical
where the edges are held together by plastic dips
and stand in the mobile phase.
 Requires no special apparatus .
 The mobile phase rises up the paper by capillary
force.
 This technique gives more constant R valacs.
2-Descending Development :
 The apparatus consists of jar provided with a
trough for the mobile phase in its upper part and
anti-siphon glass rod.

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 The sample is spotted on a starting line about 2 cm
below the anti-siphon rod and Set up in the
saturated jar for equilibration.
 After equilibration, the paper is hold with the upper end
in the solvent trough the solvent is placed in the trough
and passes upwards over antisiphon glass rod which
suspends it away the edges of the through. The other
end of the paper hangs freely in the tank.
 Development is allowed until solvent front reaches
close to the lower end of the paper
Advantages of descending development :
- Solvent flows faster
- Long distance of development can be achieved

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3-Horizontal Development :
- Shallow tank of glass or metal.
- The paper is placed horizontal on glass rods with one
end of the paper isdipped in the mobile phase.
- Sample is spotted on a line on the horizontal part of the
paper 1-2 cm from the edge.
- Solvent is flows up by capillary forces.
4- Radial Decelopment :
- The principle of this method is based on the migration of
the applied spot from the center of the paper toward
the periphery.
- Two petri-dishes tops or bottoms are used, where one
is inverted over the other
- The filter paper disc with the applied sample in the
center is stretched between the two dishes and the
mobile phase is fed through a wick-like tail from the
bottom dish to the center of the point of application.
- The solvent radiates from the center and the separated
components will be in the form of concentric rings
Advantages of Radial Development :
1- It is a fast method 2- can be demonstrated

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Detection :
This is done either by physical, chemical, biological or radiographic method.
Physical Methods:
- By colour examination for the coloured compounds.
- Flourescence detection under U.V. which gives specific
fluorescent colours.
Chemical Methods: Colourless compounds are visualized by specific spray reagents
- Aniline phthalate use reducing sugars
- Dragendorff use alkaloids
- FeCL3.phenolic use compounds
- Ninhydrin use amino acid, amino sugars
- Anisaldehyde HSO4 use terpene, sterols, sugars
Biological Methods: 1- Enzymatic Method: e.g. Detection of amylase
- The developed chromatogram is sprayed with starch
solution.
- The sprayed chromatogram is incubated for a suitable
period where amylase causes hydrolysis for the starch.
- The chromatogram is sprayed with l/KI solution.
- The amylase spots appear white on a blue background.
2- Microbial Method: e.g. Detection of antibiotics:
- The developed chromatogram is placed in a petri dish
containing the tested microorganism with the proper
nutrient medium.
- The microorganism grows over the petri dish except
where it comesin contact with the antibiotic spots.

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Radioactive Isotope Methods: 1- Auto- Radioactivity Method:
- This is done by pressing the chromatogram on an X-Ray
film in the dark allow to stand in the dark for a period of
time (overmight), the film shows dark spots on a
colourless back ground indicating the position of radio
active compounds.
2- Radio-active Chromatogram Scanner:
- This instrument automatically counts and records the
radioactivity in the chromatogram.
Uses of Paper chromatograhy :
- Identification of component mixtures
- Detection of the purity of compounds
- Major used is the separation of flavonoids and sugars.
RF VALUE :
In order to obtain a measure of the extent of movement of a
component in a paper chromatography experiment, we can
calculate an "Rf value" for each separated component in the
developed chromatogram.
The Rf value is defined as the ratio of the distance moved by the
solute (i.e. the dye or pigment under test) and the distance
moved by the the solvent where both distances are measured
from the common Origin or Application Baseline.

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