222 Chapter 4

Chromatographic Techniques
1. Introduction to chromatography, IUPAC definition of chromatography.

2. History of Chromatography-
3. Types of chromatography –
(a) Paper chromatography, (b)Thin Layer Chromatography, (c) Ion
exchange Chromatography, (d) Gas permeation Chromatography, (e)
Affinity chromatography, (g) Gas chromatography, (h) Supercritical fluid
chromatography, (i) High Performance Liquid Chromatography, (j)
Capillary electrophoresis,
4. Classification of chromatographic methods – according to separation
methods, according to development procedures.
(i)Thin Layer Chromatography: Theory and principles, outline of the
method, surface adsorption and spot shape, Comparison of TLC with other
forms of chromatography, adsorbents, preparation of plates, application of
samples, development.
(ii)Paper Chromatography- Origin, overview of technique, sample
preparation, types of paper, solvents, equilibrium, development, sample
application and detection, Identification, Quantitative methods,
applications of paper chromatography
Introduction
 Chromatography means – Colour Writing
 It is new physical technique of separation,

identification, identification and purification of
components of a mixture.
 It is used in many areas of study particularly in

chemistry, biology and medicine.
 Pigments, dyes, amino acids, vitamins, polymers, etc

can be separated by using the chromatography
technique.
 It is used for the purification and separation of organic
as well as inorganic substances.
 Found useful for the fractionation of complex mixtures,

separation of closely related compounds such as
isomers and in the isolation of unstable substances.
 IUPAC (International Union for Pure and Applied

Chemistry) defined Chromatography as -
a physical method of separation in which the
components to be separated are, distributed between
two phases, one of which is stationary phase while the
other is mobile phase, moves in a definite direction.
 Chromatography is a separation technique that uses
the size, shape, chemical properties or charge of
molecules in a sample to separate the sample into its
constituent components.
 It is often used to detect one, or a number of,
components in a complex mixture.
History of Chromatography
 The first true chromatography is usually

attributed to the Russian-Italian
botanist Mikhail Tsvet.
 Tsvet applied his observations with filter paper
extraction to the new methods of
column fractionation that had been developed
in the 1890s for separating the components
of petroleum.
 He used a liquid-adsorption column
containing calcium carbonate to separate
yellow, orange, and green plant pigments (what
are known today as xanthophylls, carotenes,
and chlorophylls, respectively).
Basics of Chromatography
TERM DEFINITION
Mobile phase or carrier solvent moving through the column

Stationary phase or
substance that stays fixed inside the column
adsorbent
Eluent fluid entering the column
Eluate fluid exiting the column (that is collected in flasks)
the process of washing out a compound through a
Elution
column using a suitable solvent
mixture whose individual components have to be
Analyte
separated and analyzed
 The analyte is loaded over the silica bed (packed in the column) and
allowed to adhere to the silica.
 Here, silica acts as the stationary phase.
 Solvent (mobile phase) is then made to flow through the silica bed
(under gravity or pressure).
 The different components of the analyte exhibit varying degrees of
adhesion to the silica and as a result they travel at different speeds
through the stationary phase as the solvent flows through it,
indicated by the separation of the different bands.
 The components that adhere more strongly to the stationary phase
travel more slowly compared to those with a weaker adhesion.
 Analytical chromatography can be used to purify compounds
ranging from milligram to gram scale.
Principle of separation of different components
 Differential affinities (strength of adhesion) of the various

components of the analyte towards the stationary and
mobile phase results in the differential separation of the
components.
 Affinity, in turn, is dictated by two properties of the

molecule: ‘Adsorption’ and ‘Solubility’.
 We can define adsorption as the property of how well
a component of the mixture sticks to the stationary
phase, while solubility is the property of how well a
component of the mixture dissolves in the mobile
phase.
 Higher the adsorption to the stationary phase, the

slower the molecule will move through the column.
 Higher the solubility in the mobile phase, the faster

the molecule will move through the column.
 So, the interplay between the above two factors determines
the differential rates at which the different components of
the analyte will move through the column.
 Adsorption and solubility of a molecule can be manipulated

by choosing the appropriate stationary phase and mobile
phase.
 Different chromatographic techniques –
(word file)
Thin layer chromatography
 Use of thin layer chromatography was first reported by two Russian

scientists, N.A Izmailov and M.S Schreiber. Later this technique was
developed further by other scientist.
 Thin layer chromatography (TLC) depends on the separation

principle.
 The separation relies on the relative affinity of compounds towards
both the phases.
 The compounds in the mobile phase move over the surface of the
stationary phase.
 The movement occurs in such a way that the
compounds which have a higher affinity to the
stationary phase move slowly while the other
compounds travel fast.
 Therefore, the separation of the mixture is attained.
 On completion of the separation process, the

individual components from the mixture appear as
spots at respective levels on the plates.
 Their character and nature are identified by suitable

detection techniques.
 The TLC plate is a thin piece of aluminium coated on one side
with the stationary phase (this phase does not move), an inert
material that does not react chemically with the sample
components.
 A mobile phase, which consists of a solvent that moves

through the stationary phase, is used to move the components
of the sample up through the stationary phase.
 A drop of the sample is placed near the bottom end of the TLC
plate. The bottom end of the TLC plate is then dipped into the
solvent (mobile phase).
 The solvent creeps slowly up the TLC plate.

 The various components of the sample move along with the
solvent at a rate that depends on how strongly they are bound
to the stationary phase.
 Strongly bound substances hardly move at all, a very weakly

bound substance may move at almost the same speed as the
solvent.
 Thus the different components (or fractions) of the sample are

separated into bands (or spots) along the length of the TLC
plate.
 This separation process is the basis of the chromatography

method
Components of Thin Layer Chromatography (TLC)
 TLC plates, preferably ready made with a stationary phase: These are stable and chemically
inert plates, where a thin layer of stationary phase is applied on its whole surface layer. The
stationary phase on the plates is of uniform thickness and is in a fine particle size.
 TLC chamber- This is used for the development of TLC plate. The chamber maintains a
uniform environment inside for proper development of spots. It also prevents the evaporation
of solvents, and keeps the process dust free.
 Mobile phase- This comprises of a solvent or solvent mixture The mobile phase used should
be particulate-free and of the highest purity for proper development of TLC spots. The solvents
recommended are chemically inert with the sample, a stationary phase.
 A filter paper- This is moistened in the mobile phase, to be placed inside the chamber. This
helps develop a uniform rise in a mobile phase over the length of the stationary phase.
Applications of Thin Layer Chromatography (TLC)
 In monitoring the progress of reactions
 Identify compounds present in a given mixture
 Determine the purity of a substance.
 Analyzing ceramides and fatty acids
 Detection of pesticides or insecticides in food and water
 Analyzing the dye composition of fibers in forensics
 Assaying the radiochemical purity of radiopharmaceuticals
 Identification of medicinal plants and their constituents

 https://www.khanacademy.org/test-
prep/mcat/chemical-processes/separations-
purifications/v/thin-layer-chromatography
 https://www.youtube.com/watch?v=lj5OWzhZSac
 https://www.youtube.com/watch?v=qdmKGskCyh8
Paper Chromatography
 Invented by Archer John Porter Martin and

Richard Laurence Millington Synge.
 Its development successfully solved the problem of

separating amino acids which are very similar to
each other.
 https://www.youtube.com/watch?v=6o0yFvDYex4
 Special papers are usually used for this technique.
 These papers should be highly purified.
 The papers used contain sufficient adsorbed water.
 Other liquids as silicone, paraffin oil are used
 Choice paper is dependent on the type of analysis
under investigation
 Whatman chromatographic papers of different types
are used.
 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.
Steps in Paper Chromatography
 Selection of Solid Support
 Selection of Mobile Phase
 Saturation of Tank
 Sample Preparation and Loading
 Development of the Chromatogram
 Drying of Chromatogram
 Detection
Applications of Paper Chromatography
 To check the control of purity of pharmaceuticals,

 For detection of adulterants,
 Detect the contaminants in foods and drinks,
 In the study of ripening and fermentation,
 For the detection of drugs and dopes in animals &
humans
 In analysis of cosmetics
 Analysis of the reaction mixtures in biochemical labs
Ion-exchange Chromatography
 Originally introduced by Sir Thompson and JT Way.
 Method was used to treat clays with the salts, resulting

in the extraction of ammonia in addition to release of
calcium
 Compounds knows as “zeolites” were introduced to
separate individual ions or electrically charged
particles.
 Synthetic resins were developed from complex ion-
exchange processes.
 https://www.youtube.com/watch?v=i4U4ndf2ayg&t=2
94s
The Applications of Ion Exchange Chromatography
 Ion exchange is the most widely used chromatographic
method for the separation and purification of charged

biomolecules such as polypeptides, proteins,
polynucleotides, and nucleic acids.
 Its widespread applicability, high capacity and simplicity,
and its high resolution are the key reasons for its success
as a separation method.
Ion exchange chromatography is widely used in several
industrial applications some of which are as follows:
 Separation and Purification of blood components such as

albumin,recombinant growth factors and enzymes.
 Biotechnology - Analytical applications such as quality
control and process monitoring
 Food and clinical research - to study wheat varieties and
the correlation of proteinuria with different renal
diseases.
 Fermentation - Cation exchange resins are used to
monitor the fermentation process during ß-galactosidase
production.
Other methods
 Gel permeation chromatography
 Affinity chromatography
 Gas chromatography
 Supercritical fluid chromatography
 Capillary electrophoresis
 High performance chromatography

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Chromatographic Techniques

1. Introduction to chromatography, IUPAC definition of chromatography.

 Chromatography means – Colour Writing

 It is new physical technique of separation,

 It is used in many areas of study particularly in

 Pigments, dyes, amino acids, vitamins, polymers, etc

 Found useful for the fractionation of complex mixtures,

 IUPAC (International Union for Pure and Applied

 The first true chromatography is usually

Mobile phase or carrier solvent moving through the column

 Differential affinities (strength of adhesion) of the various

 Affinity, in turn, is dictated by two properties of the

 Higher the adsorption to the stationary phase, the

 Higher the solubility in the mobile phase, the faster

 Adsorption and solubility of a molecule can be manipulated

 Use of thin layer chromatography was first reported by two Russian

 Thin layer chromatography (TLC) depends on the separation

 On completion of the separation process, the

 Their character and nature are identified by suitable

 A mobile phase, which consists of a solvent that moves

 The solvent creeps slowly up the TLC plate.

 Strongly bound substances hardly move at all, a very weakly

 Thus the different components (or fractions) of the sample are

 This separation process is the basis of the chromatography

 In monitoring the progress of reactions

 Identify compounds present in a given mixture

 Determine the purity of a substance.

 Analyzing ceramides and fatty acids

 Detection of pesticides or insecticides in food and water

 Analyzing the dye composition of fibers in forensics

 Assaying the radiochemical purity of radiopharmaceuticals

 Identification of medicinal plants and their constituents

 Invented by Archer John Porter Martin and

 Its development successfully solved the problem of

 Selection of Solid Support

 Selection of Mobile Phase

 Sample Preparation and Loading

 Development of the Chromatogram

 To check the control of purity of pharmaceuticals,

 Originally introduced by Sir Thompson and JT Way.

 Method was used to treat clays with the salts, resulting

 Ion exchange is the most widely used chromatographic

method for the separation and purification of charged

 Its widespread applicability, high capacity and simplicity,

 Separation and Purification of blood components such as

 Gel permeation chromatography

 Supercritical fluid chromatography

 High performance chromatography

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