PAPER CHROMATOGRAPHY

1. Paper chromatography
By
B.Nom Kumar Naik

2.  Introduction
 General procedure
 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 )
3.Developing chambers
4.Sample spotting & colour development
5.detective methods..i..e..Physical & chemical methods
6.Non – selective detective methods
 Applications of paper chromatography
 References
Contents

3. 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
Minorly “Adsorption”
S.P : - Solid ( cellulose fibres' of whatmans filter paper )
M.P : - Liquid .
Note : - Partition is more predominant than adsorption , so the principle is “ Partition”.
Introduction

4. 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.
GENERAL PROCEDURE

5. 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
component.

6. 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
one.
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.

7. Paper partition
chromatography
Paper adsorption
chromatography
Reverse phase Paper
partition chromatography
Ion exchange Paper
partition chromatography
Based on the migration forces

8. 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
adsorption chromatography
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 “.

9. Different developing techniques of paper
chromatography
Ascending paper
chromatography
Radial paper
chromatography
Descending Paper
chromatography
Ascending – descending
Paper chromatography
Developing techniques
Two dimension paper
chromatography

10. 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
upward
( 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
from above.

11. 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
chromatography
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
rings.

12. 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

13. 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
mineral contents.
Paper consist of 6% moisture.
S.P ( stationary phase )
Materials & method

14. 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

15. 1.Carbonyl paper : -
oxidation
Uses
2.Hydrophilic paper : -
Formamide Uses
Glycerol,
Glycol
Methanol
Different types of paper
Paper  Capilarity of paper  Polarity of paper
separation of polar compouds
Paper Hydrophilic compound separation of polar compouds

16. 3.Hydrophobic paper : -
Paraffine liq. uses
Note : - Here reversed phase papers are formed
4.Acetyleted paper : -
Acetylation uses
Esterification
5.Ion-exchange paper : -
uses
Paper Non-polar (S.P) Liphophilic compounds like steroids
Paper  Polarity Polar Liphophilic compounds like steroids
Paper for ion – exchange chromatography

17.  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)

18. 1) It rage between small test tubes to large jars or containers.
2) They may be made up of material like glass , steel etc.....
Developing chamber

19. 1.Spotting of the sample is done with the help of a capillary tube or automated
applicator..
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
with it.
5. At the end of the development take out the paper and mark the solvent front with
another line.
Sample spotting and development of colour

20. 6.Allow the paper to dry in drying cabinets with the provision of electrical heating
before visualisation.
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.

21.  Base on physiochemical nature
Spraying agents or detecting agents or visualizing agents
Detection
Physical Chemical
Non-specific
Specific
Auto-radioactive
method
Geiger-muller
Radioactive methodUV chamber / Fluroscence

22. 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.
Physical method
1.Radioactive method

23.  This method is used for the detection of “Florescent and Non – florescent
compounds.
 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 : -

24. 1.Iodine chamber method
2.Uv 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
reagent.
2. All the non selective methods are non destructive except universal sprayer
reagent.
Non specific or Non selective method : -

25. Sl.no Name of the compounds Spraying reagent Colour obtained
1
Phenols & Tannins Ferric chloride Blue , green , Violet
2
Amino acids Ninhydrine Pink /Purple
3
sugars Aniline-hydrogenpthalate Black grey
4
Cardiac glycosides Di-nitrobenzoic acid Orange
5
Anthraquinones Ammonia Pink
Chemical method
Specific method : -

26. Sl.no Name of the compounds Spraying reagent Colour obtained
6 Alkaloids Dragandroff reagent Brown
7 Sulphonamides
NaNO3 (0.5%) + B.M
reagent (1%) Red / Purple
8 Aldehydes & Ketones 2,4-DNP Yellow / Orange
9 Barbiturates Diphenylcarbazone Pink / Purple
10 Steroids
Antimonytrichloride in
CHCl3 Multicolours
11 Amines & Unsaturates Iodine Brown

27.  All these are selective and specific reagent in chemical method of
detection.
 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

28. 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 : -

29. 1. A text book of pharmaceutical analysis by Dr.S.Ravisankar
2. https://en.wikipedia.org/wiki/Paper_chromatography
3. https://byjus.com/chemistry/paper-chromatography/
4. https://www.britannica.com/science/paper-chromatography
5. Instrumental methods of chemical analysis by G.R.Chatwal.
6. Pharmaceutical analysis Vol – 2 by A.V.Kasture.
7. Pharmaceutical analysis by Ashutoskar.
Reference