This document provides information on paper chromatography, including its history, key terms, processes, techniques, factors that affect separation, and applications. Paper chromatography is an analytical technique used to separate mixtures based on differences in how compounds partition between a stationary phase, such as the paper, and a mobile phase, such as a solvent. Key points covered include the different types of paper chromatography, factors that influence separation like the solvent used, and how paper chromatography can be used to analyze organic compounds.

3. CONTENTS
 INTRODUCTION
 CHROMATOGRAPHY
 PAPER CHROMATOGRAPHY
 TYPES OF PAPER CHROMATOGRAPHY
 TYPES OF PAPER
 DEVELOPMENT OF CHROMATOGRAPHY
 SAMPLE SOLUTIONS
 SELECTION OF SOLVENT
 FACTOR AFFECTING Rf VALUES
 REVERSED PHASE CHROMATOGRAPHY
 SYSTEMATIC ANALYSIS
 SEPARATION OF ORGANIC COMPOUNDS BY P.C METHOD

4. Learning objectives:
 What is chromatography.
 How to make chromatograms.
 In this method we learn about different techniques of
separation which help us in different fields.
 Learn about separation of different organic solvents.

5. INTRODUCTION
CHROMATOGRAPHY is a separation technique based on the distribution of solute
between stationary phase and mobile phase.
Paper chromatography
Paper chromatography (PC) was first introduced by German scientist Christian
Schonbein Geop-pelsroeder in (1881).

6. Paper chromatography:
 PC is considered to be simplest and most widely used of chromatographic
techniques because of its applicability to isolation ,identification and
quantitative determination of organic and in-organic compounds.
1. Partition chromatography
2. Adsorption chromatography
3. ion exchange chromatography
Partition chromatography:
Partition chromatography is a liquid , liquid chromatography in which
stationary phase is a thin film of water adsorbed on a paper.
Stationary phase may be aqueous, hydrophilic and hydrophobic.
MARTIN AND
SYNGE
introduced
partition system
in 1941.

7. Mobile and stationary phase:
 Mobile phase which carries the components of the mixture through the
medium being used ,while stationary phase remain fixed in position.
 A technique used to separate and identify chemicals in a mixture.
 Relies on the movement of a gas or liquid through a medium.
In paper chromatography:
 The stationary phase is water trapped between the fibres in the paper.
 The mobile phase is a solvent
 The choice of solvent depends on what is being separated

8. The Stationary phase may be aqeous ,hydrophilic or hydrophobic in
chracter.
Aqueous stationary phase: paper holds water .it is water
equilibrated by exposing to water saturated atmosphere.Polar mixture is
separated using such paper.
Hydrophilic Stationary Phase: Hydrophilic organic solvent is
applied over the surface of paper .This solvent acts as stationary
phase.Hydrophilic solvent (usually methanol,formamide etc)
Hydrophobic Stationary phase: Hydrophbic solvents used for
stationary phase are dimethyl formamide,kerosine etc…..

10. ABSORPTION CHROMATOGRAPHY
The paper exhibits adsorption as well as a
weak ion –exchange property.Strip of filter paper ,which is made of
cellulose ,has adsorptive properties like carbon.The ions spotted on the
filter paper may be more or less adsorbed on the paper .
ION-EXCHANGE CHROMATOGRAPHY
The ion exchange papers are used for the separations of ionic
sepecies ,amino acids etc.. Ion exchage papers are either modified
forms of cellulose or a blend of cellulose with ion exchange resin.

11. TYPES OF PAPER
* Whatman No 1 filter paper
* Whatman No 4 filter paper
* Ordinary filter paper
* Modified filter paper
MAKING OF PAPERS
Whatman No:1 filter paper : It is made up of cotton cellulose
Whatman No: 4 Filter paper : It has greater thickness and fibrous in nature.it filter
quickly.
Ordinary Filter paper : It is made up of cotton cellulose .
Modified Filter paper : It is chemically prepared with carbonoxymethylcellulose.

12. DEVELOPMENT OF CHROMATOGRAMS
 It involves the separation of the mixture components when the mobile phase
is allowed to come in contact with the solute to distribute itself b/w the
mobile and stationary phase.
 REQUIRED EQUIPMENTS:
 Tank or chamber
 The solvent front is marked with a pencil line.
 drying ovens.
 spraying or detection and impregnated dishes.
 UV lamps

13. APPLICATION OF SAMPLE
The sample to be applied is dissolved in the mobile phase and applied as a small
spot on the origin line,using capillary tube or micropipette.
Very low concentration is used to avoid large zone
* The spot is dried on the filter paper and is placed in developing chamber.

14. TYPES OF DEVELOPING chromatograms
 Ascending-Descending Paper Chromatography : It 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.
 Radial Paper Chromatography : It is also called Circular Chromatography.
Here a circular filter paper is taken and the sample is deposited at the center
of the paper.
 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 right angle to the direction of the first run.

15. Circular / Radial
chromatograms
Two-way
chromatograms
ASCENDING-
DESCENDING

16.  Descending Paper Chromatography (a downward slop): In this type,
development of the chromatogram is done by allowing the solvent to travel
down the paper.
 This is carried out in a special chamber where the solvent holder is at the top
and the solvent flows down the paper.
 Advantage is that ,development is faster.
 Ascending Paper Chromatography(a upward slop) : Here the solvent travels
up the chromatographic paper. Both Descending and Ascending Paper
Chromatography are used for the separation of organic and inorganic
substances.

17. DETECTION OF SEPARATED SUBSTANCE
 1)Destructive techniques
 Specific spray reagent ,samples destroyed before detection e.g. –
ninhdrin reagent
 2)Non-destructive techniques
 For radio active materials- Geiger Muller counter
 Uv chamber,iodine chamber

18. CHOICE OF THE SOLVENT FOR CHROMATOGRAPHY
 The commonly employed solvents are the polar solvents, but the choice
depends on the nature of the substance to be separated
 If pure solvents do not give satisfactory separation, a mixture of solvents of
suitable polarity may be applied.

19. Quantitative and qualitative analysis:
 Qualitative Analysis : refers to analyses in which substances are identified or
classified on the basis of their chemical or physical properties, such as
chemical reactivity, solubility, etc.
 Quantitative Analysis : refers to analyses in which the amount or
concentration of an analyte may be determined (estimated) and expressed as
a numerical value in appropriate units.

20. Rf values an solvents:
The retention factor (Rƒ) may be defined as the ratio of the distance
traveled by the substance to the distance traveled by the solvent.
 The formula is:
Rf = distance moved by sample
distance moved by solvent
The answer is never greater than 1.

21. FACTORS AFFECTING Rf VALUES
 TEMPERATURE
 PAPER
 SIZE OF TANK

22. REVERSED PHASE CHROMATOGRAPHY
 The sparingly soluble compound in water moves absolutely with the solvent
front thus no separation can occur on the paper while doing the
chromatography.
 The paper in such case is specially impregnated with a non-aqueous medium
,which serves as stationary phase .
 Mobile phase on the other hand ,is kept more polar and the technique is
called REVERSED PHASE CHROMATOGRAPHY.
The substance which is
used for stationary phase
is olive oil, silicon
oil,rubber etc

23. Organic compound
An organic compound is virtually any chemical compound that contains
carbon
Lower classifications
Hydrocarbon
Protein
Alcohol
Ester
Amine
Aldehyde
Ketone
Ether
Thiol
Oil
Amino alcohol
Organometallic compounds

24. Separation of Organic Compounds by Paper Chromatography:
Alcohol:
METHOD:
volatile alcohol are not directly chromatographed rather they are converted
into suitable derivative before chromatography . Higher aliphatic alcohols are
not volatile and they must be separated by reverse – phase chromatography .
Derivatives :
(a) 3,5 di nitro benzoates
(b) Xanthates of the most lower aliphatic alcohols (e.g methyl- ,ethyl- , propyl- ,
butyl- ,…….., etc ).

25. Aldehyde and ketones:
Derivatives : Aliphatic and alicyclic aldehydes and Ketones are
used as derivatives in chromatography as 2,4-dinitrophyenylhydrazone
and paced on paper previously treated with 25% solution of
dimethylformamide for development with cyclohexane.
Spots colour : The spot are usually orange to red and therefore can
be easily detected.The intensity of the spot can be increased in visible
light by spraying 1% ethanolic sodium hydroxide.The spots turn violet.

26. AMINES:
 Method : Paper chromatography is used for lower dialiphatic amines,
amino alcohols and high amines.However the higher amines in the free base
from involves reversed phase chromatography for resolution .

Chemical solvent : n-butanol-ethanol-acetic acid and water is used as
chemical solvent.

Locating agent: ninhydrin is used as locating agent for lower aliphatic
amines and amino alcohols.

27. APPLICATIONS
 Separation of mixtures of drugs.
 Identification of medicine.
 Identification of impurities.
 Analysis of metabolites of drugs in blood ,urine…
 Separation of organic solvents like vitamins and proteins etc

28. REFERENCES
 From the David Harvey (Modern analytical chemistry)Edition 6th chapter
12.
 From the Vogels book(Quantitative chemical Analysis)Edition 5th Chapter7.
 From the website https://www.chemguid.com

29. SUMMARY:
 Paper chromatography with Ions exchange and Absorption chromatography
techniques are very helpful in separation of ionic species and organic solvents
,which will help us a lot in different fields for separation.
 This technique is widely used in industrial level or in small scale,for
separation of different medicine,or as a dyes for dying clothes.

30. ANY QUESTION???????????