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B.Nom Kumar Naik
Types of Paper chromatography
Difference between partition & adsorption chromatography
Different development techniques of paper chromatography
Materials & methods
1.Stationary phase ( S.P )
2. Mobile phase ( M.P )
4.Sample spotting & colour development
5.detective methods..i..e..Physical & chemical methods
6.Non – selective detective methods
Applications of paper chromatography
Definition : - Paper chromatography is a chromatography technique used to separate
mixture of chemical substances into its individual compounds.
It is an inexpensive method of separating dissolved chemical substances by their different
migration rates across the sheets of paper.
It is a powerful analytical tool that uses very small quantities of material. Paper
chromatography was discovered by Synge and Martin in the year 1943
Principle : - Majorly “Partition”
S.P : - Liquid ( whatmans filter paper consisting of cellulose layer which contain liquid )
M.P : - Liquid
S.P : - Solid ( cellulose fibres' of whatmans filter paper )
M.P : - Liquid .
Note : - Partition is more predominant than adsorption , so the principle is “ Partition”.
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.
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.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
(M.P). 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 :
1.The polarity of the sample molecule. The non polar components travel faster than the polar
2.The attraction between the sample molecule and the solvent or solvent mixture.
3.The attraction between the sample and the silica.
Suppose any sample compound mixture contains three colored molecules green, blue and red.
According to their polarity, the order of these compounds is green<blue<red. Thus the most non polar
green will travel first along with the mobile phase. Then blue and at last most polar compound the red
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
Reverse phase Paper
Ion exchange Paper
Based on the migration forces
Paper partition chromatography
1.S.P : - LIQUID (Present in the pores of the
cellulose fibres in the filter paper )
2.M.P : - LIQUID (single / mixture of the solvent )
3. Migration of the substance id due to difference
in “PARTITION Coefficient “.
Difference between paper partition chromatography and paper
Paper adsorption chromatography
1.S.P : - SOLID (Silica gel)
2.M.P : - LIQUID (Single / mixture of the solvent )
3.Migration of the substance is due to
“ADSORPTIVE Power “.
Different developing techniques of paper
Ascending – descending
Two dimension paper
Ascending paper chromatography
Here the solvent travels up the
chromatographic paper. Both descending and
ascending paper chromatography are used for
the separation of organic and inorganic
substances. The sample and solvent move
( Against gravitational force ).
Descending paper chromatography
Development of the chromatogram is done
by allowing the solvent to travel down the
paper. Here, mobile phase is placed in
solvent holder at the top. The spot is kept at
the top and solvent flows down the paper
Ascending-descending paper chromatography
This is the hybrid of both of the above
techniques. The upper part of ascending
chromatography can be folded over a rod in
order to allow the paper to become descending
after crossing the rod.
Circular chromatography paper
A circular filter paper is taken and the
sample is deposited at the centre of the
paper. After drying the spot, the filter
paper is tied horizontally on a Petri
dish containing solvent, so that the wick
of the paper is dipped in the solvent. The
solvent rises through the wick and the
components are separated into concentric
Two-dimensional paper chromatography
In this technique a square or rectangular paper is used. Here the sample is applied to
one of the corners and development is performed at a right angle to the direction of
the first run
Selection criteria for materials and method : -
1. Qualitative / Quantative analysis .
2. Analytical / preparative chromatography.
3. Hydrophilic / Lipophilic compounds.
Whatsman grade filter paper obtained from purified cotton fibre.
It consist of 99% -cellulose and remain 1% -cellulose,pentose and
Paper consist of 6% moisture.
S.P ( stationary phase )
Materials & method
Sl.no Whatsman grade Type of paper used Developing time of colour
1 Grade – 1 Slow / standard paper 15 – 16 min
2 Grade - 3
Preparative paper for
preparative chromatography Exact 5 min
3 Grade – 4 Fast paper 9 min
4 Grade – 31T Very fast paper with in 5 min
Different grades of papers
1.Carbonyl paper : -
2.Hydrophilic paper : -
Different types of paper
Paper Capilarity of paper Polarity of paper
separation of polar compouds
Paper Hydrophilic compound separation of polar compouds
3.Hydrophobic paper : -
Paraffine liq. uses
Note : - Here reversed phase papers are formed
4.Acetyleted paper : -
5.Ion-exchange paper : -
Paper Non-polar (S.P) Liphophilic compounds like steroids
Paper Polarity Polar Liphophilic compounds like steroids
Paper for ion – exchange chromatography
It should be more polar than S.P.
It may be single / mixture of solvents.
It should not react with components and spraying reagents.
There are two types of M.P
1.Hydrophilic mobile phase : -
eg : - 1. n-butanol : Glacial acetic acid : water (4 : 1 : 5 )
2. Isoproponal : Ammoia : water ( 9 : 1 : 2 )
2.Hydrophobic mobile phase : -
eg : - Cyclohexane , Diethyl ether
NOTE : -
If the compound is polar we can use Chloroform as solvent ( M.P ).
If the compound is medium polar we can use Benzene as solvent.
If the compound is low polar we can use Cyclohexane as solvent.
M.P ( Mobile phase)
1) It rage between small test tubes to large jars or containers.
2) They may be made up of material like glass , steel etc.....
1.Spotting of the sample is done with the help of a capillary tube or automated
2.The sample is applied as a neat spot on a horizontal line drawn with a pencil close
to one edge.
3. Allow the spot to dry and then immerse the paper in the developing chamber as per
the selected technique with the marked spot above the solvent level.
4.The solvent begins to move and draws the sample components differentially along
5. At the end of the development take out the paper and mark the solvent front with
Sample spotting and development of colour
6.Allow the paper to dry in drying cabinets with the provision of electrical heating
Note : -
1. The spotting diameter should be in range between 3 – 4 mm.
2. For the loading of sample above edge or base line i.e..
for Ascending paper chromatography 4 – 5 cm
for Descending paper chromatography 8 – 9 cm.
3. The distance between 2 spots is 3 – 4 cm.
Base on physiochemical nature
Spraying agents or detecting agents or visualizing agents
Radioactive methodUV chamber / Fluroscence
A.Geiger-muller method : -
Chromatogram was placed in GMC ( geiger-muller chamber ). And it is used
for the identification of radioactivity.
B.Autoradioactive method : -
Chromatogram is pressed between glass slide and x – ray slide , from these we
can identify the quantity of a compound.
This method is used for the detection of “Florescent and Non – florescent
Non – florescent compounds can be identified at 256 rpm .
Florescent compounds produce different colours like “ Green , Blue , Violet “
with a background of Black colour.
Non – florescent compounds produce “ Black colour “ with a background of
different colours like Blue, Green , Violet .
2.UV chamber method : -
1.Iodine chamber method
3.Universal sprayer reagent ( sulphuric acid )
Note : -
1. All the selective methods are destructive methods including universal sprayer
because they destroy the original compound and gives colour when we spray
2. All the non selective methods are non destructive except universal sprayer
Non specific or Non selective method : -
Sl.no Name of the compounds Spraying reagent Colour obtained
Phenols & Tannins Ferric chloride Blue , green , Violet
Amino acids Ninhydrine Pink /Purple
sugars Aniline-hydrogenpthalate Black grey
Cardiac glycosides Di-nitrobenzoic acid Orange
Anthraquinones Ammonia Pink
Specific method : -
Sl.no Name of the compounds Spraying reagent Colour obtained
6 Alkaloids Dragandroff reagent Brown
NaNO3 (0.5%) + B.M
reagent (1%) Red / Purple
8 Aldehydes & Ketones 2,4-DNP Yellow / Orange
9 Barbiturates Diphenylcarbazone Pink / Purple
11 Amines & Unsaturates Iodine Brown
All these are selective and specific reagent in chemical method of
Universal spraying reagent is “Sulphuric acid”( 4 ml sulphuric acid +
96 ml ethanol ).
Modified papers should be taken to spray Sulphuric acid (Universal
sprayer) because it is not suitable for paper chromatography and
It will be suitable for modified
papers like glass fibres paper
After staining the reagent ( Sulphuric acid ) the paper will appear
as Black colour
1) There are various applications of paper chromatography. Some of the uses of
Paper Chromatography in different fields are discussed below:
2) To study the process of fermentation and ripening.
3) To check the purity of pharmaceuticals.
4) To inspect cosmetics.
5) To detect the adulterants.
6) To detect the contaminants in drinks and foods.
7) To examine the reaction mixtures in biochemical laboratories.
8) To determine dopes and drugs in humans and animals
Paper Chromatography Applications : -
1. A text book of pharmaceutical analysis by Dr.S.Ravisankar
5. Instrumental methods of chemical analysis by G.R.Chatwal.
6. Pharmaceutical analysis Vol – 2 by A.V.Kasture.
7. Pharmaceutical analysis by Ashutoskar.