Mr.

RANJEET RANJANJA
 Contents
 Introduction

 Principle of Separation

 Modes of Paper Chromatography

 Experimental Details for Qualitative Analysis

 Experimental Details for Quantitative Analysis

 Applications

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 Introduction
 The most dramatic advance in the history of chromatography
took place in 1944.

 It was invented by two British biochemists, Archer John

Porter Martin and Richard Laurence Millington Synge.

 In 1941 Martin and Synge began working together on

proteins, which are made up of chains of amino acids.

 The duo was trying to characterize a particular protein by

determining the precise numbers of each amino acid present.

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 Amino acids are so similar to each other, however, that the
problem of separating them had defeated a whole generation of
biochemists.

 Martin and Synge's development of paper

chromatography to solve this problem was an instant success.

 It worked not only on amino acids but also on various other

mixtures. The two scientists were awarded the 1952 Nobel
Prize in chemistry for their work.

 Synge determined the structure of an antibiotic peptide called

"Gramicidin-S.

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 Frederick Sanger used paper chromatography to figure out
the structure of the insulin molecule. He determined the
number of amino acids in it as well as the order in which they
occurred.

 Calvin discovered the complex series of reactions that enable

green plants to convert solar energy into the chemical
energy stored in food using paper chromatography in 1950s.

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 It was also used by Austrian-American biochemist Erwin
Chargaff, who modified the technique to study the
components of the nucleic acid molecule. His research
revealed four components, or nitrogenous bases, that occur in
pairs.

 British biochemists James Dewey Watson and Francis Harry

Compton Crick later used these results to work out the
structure of DNA (deoxyribonucleic acid).

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There are two types of paper chromatography, they are:

PAPER ADSORPTION CHROMATOGRAPHY

Paper impregnated with silica or alumina acts as adsorbent
(stationary phase) and solvent as mobile phase.

PAPER PARTITION CHROMATOGRAPHY

Moisture/ Water present in the pores of cellulose fibers
present in filter paper acts as stationary phase and solvent
used as mobile phase.

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 PRINCIPLE OF SEPARATION
 The principle involved is partition chromatography wherein
the substances are distributed or partitioned between liquid
phases. One phase is the water, which is held in the pores of
the filter paper used; and other is the mobile phase which
moves over the paper.

 The compounds in the mixture get separated due to

differences in their affinity towards water (in stationary
phase) and mobile phase solvents during the movement of
mobile phase under the capillary action of pores in the paper.
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Principle of separation Cont.,

 The principle can also be adsorption chromatography between

solid and liquid phases, wherein the stationary phase is the
solid surface of paper and the liquid phase is of mobile phase.

 But most of the applications of paper chromatography work on

the principle of partition chromatography, i.e. partitioned
between to liquid phases.

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 Modes of Paper chromatography

Based on the way the development of chromatogram on paper

is done in procedures, we have, broadly, five modes of
chromatography.
1. Ascending
2. Descending
3. Ascending-Descending
4. Circular/ Radial
5. Two-Dimensional

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1. Ascending Technique

When the development of the paper is done by allowing the solvent

to travel up the paper, it is known as ascending technique.

 The chromatogram of this technique

ascends slowly due to the mobile phase
movement in a upward direction.

 The solvent is kept at the bottom of the

filter paper or stationary phase with the
end of the filter paper dipped in.

 The component mixture spot is kept

well above the solvent level and is not
allowed to touch the spot.

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2. Descending Technique

When the development of the paper is done by allowing the solvent

to travel down the paper, it is known as descending technique.

The mobile phase in this ADVANTAGE:

Development is faster
type is kept at the top of
the chromatogram and the
components of the mixture
separates out downward
due to gravity and
capillary action of the
filter paper.
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3. Ascending-Descending Technique

 A hybrid of above two techniques is called ascending-descending

chromatography.

 Only length of separation increased, first ascending takes place

followed by descending.

 Overall shows a bi-directional movement of the mixture

components.

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4. Circular/ Radial Technique
In this type of paper chromatography the solvent moves from the
centre towards the peripheral regions of the filter paper. The
radiating mixture component is allowed to spread till all the
components have separated out. For precaution the entire system is
covered with the help of a Petri dish.
The centre of the paper is allowed to be dipped into the solvent
and the coloured components radiates out in concentric circles.

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5. Two- Dimensional Technique
 The chromatogram in this type
develops at right angle to each other
and the filter paper is dipped at right
angle once the first chromatogram is
complete.
 The second chromatogram then
develops at right angle to the first
one.

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Experimental details for qualitative analysis

1. Choice of Development Technique

2. Choice of Stationary Phase

3. Proper Developing Solvent System

4. Preparation of Sample

5. Application of Sample (Spotting)

6. Drying the Chromatogram

7. Detecting or Visualizing

8. Calculation of Rf Value
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1. Choice of Development Technique

The choice of technique depends upon the nature of the

substances to separated.

 Ascending
 Descending
 Ascending-Descending
 Circular/ Radial
 Two-Dimensional

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2. Choice of Stationary Phase

 Chromatography makes use of paper which acts as a

stationary phase.
 Paper essentially consists of cellulose fibers which are
polymers having -OH functional groups sticking out of the
polymer chains.
 These groups lead to retention and separation of surface
absorbed molecules.
 In practice the separating molecules equilibrate between the
layer of adsorbed water and the mobile phase solvent.

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 2. Choice of Stationary Phase Cont.,

The choice of paper is dependent on the type of problem under

investigation. The prime factors, that govern the choice, are as
follows:
 Whether the paper is being used for quantitative or
qualitative analysis;
 Whether it is used for analytical or preparative
chromatography; or
 Whether the substances used are hydropilic or lipophilic,
neutral or charged species.

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 2. Choice of Stationary Phase Cont.,

Various types of Whatmann papers are available.

Rate of Flow
Fast Medium Slow
Thin Paper No. 4 No. 7 No. 2
No. 54 No. 1 No. 20
No. 540
Thick No. 31 No. 3
Paper No. 17 No. 3 MM

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 2. Choice of Stationary Phase Cont.,

 Whatmann filter papers commonly used for chromatographic

purposes have a content of 99% of α-cellulose. The rest is
mineral content.
 For the efficient separation of polar substances, the exchange
capacity of the paper is increased by increasing the carboxyl
content (1.4%) by partial oxidation.
 It is possible to increase the capillarity of the paper by partial
hydrolysis which is achieved by soaking the filter paper for 24 h
in 7% hydrochloric acid and washing successfully with water
and ethanol.
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 2. Choice of Stationary Phase Cont.,

 Cellulose paper is also used as a support for various adsorbents

like alumina, silica, zirconium oxide etc. which get precipitated
in the pores of the filter paper to produce a thin sheet of the
adsorbent with the flexibility of the paper but having adsorbent
characteristics of the precipitate.
 Chelating agents such as 8-hydroxyquinoline, dimethyl
glyoxime and several other compounds impart papers with
special characteristics.
 Papers have also been impregnated with powdered or liquid ion
exchangers to produce ion exchange papers.
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 2. Choice of Stationary Phase Cont.,

Modified Papers for Paper Chromatography

Type Mobile Phase
Carboxyl papers Cationic separation of protonated amines
and amino acids

Acetylated Papers RP chromatography of lipophilic substances

like steroids, insecticides and pigments and
also metal cations.
Kieselguhr papers, Separation of low polarity substances such as
Alumina papers, amines, fatty acids, steroids, triglycerides,
Zirconia papers, Silica vitamins and pesticides
papers

Ion Exchange Papers Ion Exchange paper chromatography

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 3. Proper Developing Solvent System

Criteria for selection of a working solvent system:

 Solvents should not be toxic or carcinogenic. In such cases

adequate handling care should be exercised.
 The solvent mixture composition should not change with
time.
 Solvent constituents should not chemically react with any of
the sample constituents.
 Solvents should not interfere with the detection of spots on
the developed paper.
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 Differences in Rf values of any two components should be at
least 0.05 for differentiation between two closely spaced spots.
 The Rf values of the sample should lie between 0.05 and 0.85
in the system.
Suitable solvent systems for paper chromatography

Water as a stationary Phase

– Isopropanol: ammonia: water [9:1:2]
– N-butanol: glacial acetic acid: water [4:1:5]
– Phenol saturated with water

SP (dimethyl ether):- Cyclohexane

SP (kerosene) :- 70% isopropanol
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3. Proper Developing Mobile Phase cont.,

Commonly used solvent combinations:

 Strongly polar compounds:
o ethyl acetate: n-butanol: acetic acid: water - 80:10:5:5

 Polar compounds:
o 10% methanol or less in dichloromethane

 Strongly basic compounds:

o 10% ammonium hydroxide in methanol and make 1-10%
mixture of this in dichloromethane

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 4. Preparation of Sample

 Pure solutions can be applied directly on the paper but solids

are always dissolved in small quantity of a suitable solvent.

 Biological tissues are treated with suitable solvents and their

extracts obtained.

 The sample volume of 10-20 µL having as many µg of the

substances is that ideal quantity to be spotted.

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 5. Application of Sample (Spotting)

Preparation of Paper
 Cut the paper into desired
shape and size depending upon
work to be carried out.

 The starting line is marked on

the paper with an ordinary
pencil 2 cm from the bottom
edge.

 On the staring line marks are

made 2 cm apart from each
other.

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Spotting:

 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 larger zone.

 The sample is applied as a neat spot on a horizontal line drawn

with a pencil close to one edge.

 The spot is dried on the filter paper and is placed in developing

chamber.

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Developing:

 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.

 The solvent begins to move and draws the sample components

differentially along with it.

 At the end of the development take out the paper and mark the
solvent front with another line.

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 6. Drying of Chromatogram

They are dried by cold or hot air depending on volatility of

solvents. A simple hair dryer is a convenient device to dry
chromatograms.

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 7. Detecting or Visualizing

 Coloured spots are easily observed on developed

chromatograms.
 However, different approaches need to be adopted when
colourless components are to be observed.
 It is convenient to classify such methods as specific or
non-specific.

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Non-specific methods (Physical):
 Iodine chamber: The developed plate is suspended in a
closed jar containing a few crystals of iodine for about a
minute. In presence of iodine vapour most organic
compounds appear as brown spots.
 UV viewing cabinet:
 Majority of colourless compounds can be viewed under
illumination with UV light in a UV viewing cabinet.
 Commonly the cabinets are equipped with a long
wavelength (366 nm) and short wavelength (254 nm)
light sources.
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 Specific methods (Chemical methods)

• Ferric chloride • Phenolic comp. & tannins

• Ninhydrin in acetone • Amino acids
• Dragendroff’s reagents • Alkaloids
• 3,5-dinitro benzoic acid • Cardiac glycosides
• Molisch reagent • Carbohydrates

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 8. Calculation of Rf VALUE

Rf VALUE (Retardation Factor)

The Rf defines the movement

of the substance relative to
the solvent front in a given
chromatographic system.

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 Factors affecting Rf VALUE

 The temperature

 The purity of the solvents used

 The quality of the paper, adsorbents & impurities present in

the adsorbents

 Chamber saturation techniques, method of drying &

development

 The distance travelled by the solute & solvent

 Chemical reaction between the substances being partitioned.

 pH of the solution
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 Rx VALUE
 In many cases it has been observed that the solvent front is run
off the end of the paper.

 It is the ratio of distance travelled by the sample and the

distance travelled by the standard.

 Rx value is always closer to 1.

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Experimental details for Quantitative Analysis

For quantitative analysis, the preliminary separation is done

in the same way as in qualitative analysis. Then, the assay
can be performed either after extraction from the paper or
in situ on the paper.

1. Estimation after Extraction from the paper

2. In situ methods

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1. Estimation after Extraction from the paper
In this technique, the spots are cut into portions and eluted with
solvents. This solution can be analyzed by any techniques of
analysis like gravimetric estimation, spectrophotometry,
Colorimetry, Polarography, Flame photometry, etc.

In the assessment of the merits of any procedure, the following

information is required:
 The nature of the substance to be assayed.

 The scientific equipment available and its sensitivity.

 The time available and

 The alternative method, available, if any, and their relative

accuracies
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 2. In situ methods
 By Visual Assessment: The simplest procedure is to see the spot by the
human eye. However, it is not very accurate.

 By Measurement of areas:

 if the outline of the spots or zones are well defined, the size of the
spot (length or areas) may serve for determining the quantity of the
substances.

 Then a linear relationship is obtained between the spot area and

amount of the substance present.

 The random errors in the procedure are generally high as a results of

the variations of the spot shape during separation.

 Other drawbacks: Volume of applied sample and speed of

application
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2. In situ methods cont.,

 By Densitometer: It is a method whereby the intensity of the colour

of a substance is measured directly on the chromatogram.

 By Potentiometer: Changes in the potential of a metal electrode in

contact with the filter paper is also utilised with quadrant
electrometer or electronic voltmeters.

 Fluorimetry: The compound to be determined by fluorimetry must

be fluorescent or convertible into fluorescent derivatives.

 Radiotracer Method: The compound containing radioactive

element is labeled and treated with locating reagent. Using Geiger
Muller counter.

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 SOURCES of ERROR
 Error during application of the spots:

o Apply minimum volume of the concentrated solution in order to

avoid diffusion through the paper which leads to poor separation

o Spots should be approximately of the same diameter.

 Development:

o Improper adjustment of the paper in the tank leads to this error

so the paper should be held vertically.

o Do chamber saturation
 Detection:

o The spraying methods affect the final result

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 APPLICATIONS
Innumerable applications have been reported in analysis of
different classes of compounds such as:
 Amino acids and organic acids

 Alkaloids

 Polysaccharides

 Proteins and peptides

 Natural and artificial pigments

 Inorganic cations

 Plant extracts

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Applications cont.,

Typical applications in key areas are briefly outlined here:

1. Reaction monitoring:
In a chemical reaction over a period of time the concentration of
reactants decreases whereas the concentration of products increases.
Developing the chromatogram over different time intervals by spotting
the reactants can give a fair idea on the progress of reaction.
Traditionally the technique was used for qualitative monitoring but
availability of densitometers made quantitative estimations possible.

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Applications cont.,

2. Isolation & Purification:

Paper chromatography has been put to use as a purification and

isolation technique for components of mixtures. The separated
components on the paper are cut, dissolved in suitable solvents and
their absorption characterised at specific wavelengths using
spectrophotometric methods.

3. Foods & Beverages:

Paper chromatography has been used for analysis of food colours in
synthetic drinks and beverages, ice creams, jams & jellies, sweets, etc.

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Applications cont.,

2. Isolation & Purification:

Paper chromatography has been put to use as a purification and

isolation technique for components of mixtures. The separated
components on the paper are cut, dissolved in suitable solvents and
their absorption characterised at specific wavelengths using
spectrophotometric methods.

3. Foods & Beverages:

Paper chromatography has been used for analysis of food colours in
synthetic drinks and beverages, ice creams, jams & jellies, sweets, etc.

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Applications cont.,

4. Forensics:

The major applications are for identification and comparison against

reference standards for drugs and their metabolites in viscera, explosive
residues from blast sites, inks used in forgery of documents and paint
pigments investigations in hit and run road accident cases.
5. Pharmaceuticals:
It offers a cost-effective alternative to monitoring the active ingredients
in drug forms administered in bioanalytical studies. Its main
contribution is when the quantity of sample available is in minute
amounts.

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