of

Chromatograp
hy
Prepared By: Mr. Ravi Somabattini
Hall ticket no. :13M71R0054
Class : Final B. Pharmacy
Chromatography
Chromatography is a method of separation in which the
components to be separated are distributed between two
phases, one of these is called a stationary phase and the other is
a mobile phase which moves on stationary phase in a definite
direction. The component of the mixture redistribute
themselves between two phases by a process which may be
adsorption, partition, ion exchange or size exclusion.
The stationary phase can be solid or a liquid and the mobile
phase can be liquid, gas or a supercritical fluid.
Illustration of Chromatography

Separation

Mobile Phase

Mixture Components
 Examples of Chromatography

Thin Layer Chromatography Paper Chromatography is an Column Chromatography in Chemistry

(TLC) is a chromatography analytical method that is used to is a method use to purify individual
technique used to separate non- separate coloured chemicals or chemical compounds from a mixtures of
volatile mixtures. Thin layer substances, especially pigments. This compounds. It is often used for preparative
chromatography is performed can also be used in ink experiments. applications on scale from micrograms up
on a sheet of glass, plastic or to kilograms.
aluminium foil, which is coated
with the thin layer of adsorbent
material, usually silica gel,
aluminium oxide, or cellulose.
 Examples of Chromatography

Ion Exchange Chromatography (Ion Chromatography) is a Size-Exclusion Chromatography (SEC) is a

process that allows the separation of ions and polar molecules chromatographic method in which molecules in a solution
based on their affinity to the ion exchanger. It can be used for are separated by their size, and in some cases molecular
almost any kind of charged molecules including large protein, weight. It is usually applied to large molecules or
small nucleotide and amino acids. The solution to be injected is macromolecular complexes such as proteins and industrial
called Sample and individually separated components are called polymers.
analytes.
Introduction
The Term Chromatography (chroma = a colour; graphein = to
write) is the collective term for a set of laboratory techniques
for the separation of mixtures.
Chromatography involves a sample (or sample extract) being
dissolved in a mobile phase (which may be a gas, a liquid or a
supercritical fluid).
The mobile phase is then forced through an immobile,
immiscible stationary phase.
The phases are chosen such that components of the sample
have differing solubilities in each phase.
A component which is quite soluble in the stationary phase will
take longer to travel through it than a component which is not
very soluble in the stationary phase but very soluble in the
mobile phase.
As a result of these differences in mobilities, sample
components will become separated from each other as they
travel through the stationary phase.
Techniques such as H.P.L.C. (High Performance Liquid
Chromatography) and G.C. (Gas Chromatography)
use columns - narrow tubes packed with stationary phase,
through which the mobile phase is forced.
The sample is transported through the column by continuous
addition of mobile phase. This process is called elution.
History
The subject of Chromatography was introduced into scientific
world in a very modest way by M. Tswett in 1906.
He employed a technique to separate various pigments such as
chlorophylls and xanthophylls by passing the solution of these
compounds into the glass column which was packed with
finely divided calcium carbonate.
After the later, Thompson and Way had realized the Ion
Exchange properties of soils.
Almost after three decades, in 1935 Adams and Holmes
observed the Ion Exchange characteristics in crushed
phonograph. This observation opened the field for preparation
of Ion Exchanged resins.
The concept of Gas-Liquid Chromatography was first
introduced by Martin and Synge in 1941.
They were also responsible for the development in Liquid-
Liquid chromatography.
In 1944, from Martin laboratory, the separation of amino acid by
paper chromatography was reported.
In 1952, the importance of the chromatography was observed
when both Synge and Martin were awarded with Nobel Prize.
In 1959, a technique known as Gel Filtration chromatography
was observed which is used to separate low molecular weight
substances from high molecular substances.
In 1960, further improvement in liquid chromatography led to the
development of High Performance Liquid Chromatography.
The following decade of 1970’s saw an improvement in the field
of adsorption chromatography in the form of Affinity
chromatography which was mainly based on biological
interactions.
A new field was originated which was supercritical fluid
chromatography.
Supercritical fluid chromatography is a hybrid of gas and
liquid chromatography and combine advantageous feature of
the both gas and liquid chromatography.
It will not be wrong to say that the entire twentieth century can
be named as the century of chromatography.
 Classification Of Chromatography

On the basis of interaction of On the basis of On the basis of physical

solute to the stationary phase chromatographic bed shape state of mobile phase

Liquid Super Critical

Two Three
Chromatograp Fluid
Adsorption Ion Exchange Dimensional Dimensional
hy Chromatograp
Chromatograp Chromatograp
hy
hy hy
Gas
Chromatograph
Partition Column y
Size Exclusion Chromatography
Chromatograp
Chromatography
hy

Paper
Thin Layer
Chromatograph
Chromatography
y
On the basis of interaction of
solute to the stationary phase
 Adsorption Chromatography
 Partition Chromatography
 Ion Exchange Chromatography
 Size Exclusion Chromatography
Adsorption Chromatography
Definition:
Adsorption chromatography is probably one of the
oldest types of chromatography around. It utilizes a mobile
liquid or gaseous phase that is adsorbed onto the surface of a
stationary solid phase. The equilibration between the mobile
and stationary phase accounts for the separation of different
solutes.
Principle:
Principle of Adsorption Chromatography involves competition
of components of sample mixture for active site on adsorbent.
These active sites are formed in molecule due to
 Cracks
 Edges
Separation occurs because of the fact that an equilibrium is
established between molecules adsorbed on stationary phase
and those which are flowing freely in mobile phase.
The more the affinity of the molecule of particular component,
less will be its movement.
Types:

Adsorption Chromatography

Column Thin Layer

Gas Solid
Chromatograph Chromatograph
Chromatography
y y
Partition Chromatography
Definition:
This form of chromatography is based on a thin
film formed on the surface of a solid support by a liquid
stationary phase. Solute equilibrates between the mobile phase
and the stationary liquid.
Principle:
Separation of components of a sample mixture occurs because of
partition. Stationary phase is coated with a liquid which is
immiscible in mobile phase.

Partition of component of sample between sample and liquid/ gas

stationary phase retard some components of sample more as
compared to others. This gives basis for separation.

The stationary phase immobilizes the liquid surface layer, which

becomes stationary phase. Mobile phase passes over the coated
adsorbent and depending upon relative solubility in the coated
liquid, separation occurs. The component of sample mixture
appear separated because of differences in their partition
coefficient.
Types:

Partition Chromatography

Liquid-liquid Gas-liquid
Chromatography Chromatography
Ion Exchange Chromatography
Definition:
Ion Exchange Chromatography (Ion
Chromatography) is a process that allows the separation of
ions and polar molecules based on their affinity to the ion
exchanger. It can be used for almost any kind of charged
molecules including large protein, small nucleotide and amino
acids. The solution to be injected is called Sample and
individually separated components are called analytes. It is
often used in protein purification, water analysis, and quality
control.
Principle:
Ion Exchange Chromatography is based on the
relative retention of the ions during their progress
through an ion exchange column which has
functional group of opposite charge attached to its
surface. The stronger the charge on the ion, the
greater is the retention time in the column.

Ion chromatography is used to separate organic or

inorganic charged substances. The stationary
phases used are based on typical ion exchange
resins.
 Ion Exchange
Types: Chromatography

Cation Exchange Anion Exchange

Chromatography Chromatography

Solute cations are attached to Solute anions are attached to

the negatively charged sites the positively charged sites
covalently bond to the stationary covalently bond to the
phase stationary phase
Size Exclusion Chromatography
Definition:
Size-Exclusion Chromatography (SEC) is a
chromatographic method in which molecules in a solution are
separated by their size, and in some cases molecular weight. It
is usually applied to large molecules or macromolecular
complexes such as proteins and industrial polymers.
Typically, when an aqueous solution is used to
transport the sample through the column, the
technique is known as gel-filtration
chromatography, versus the name gel-
permeation chromatography, , which is used
when an organic solvent is used as a mobile
phase.
Size Exclusion
Chromatography
 Principle:
 A mixture of molecules dissolved in liquid (the mobile phase) is
applied to a chromatography column which contains a solid support
in the form of microscopic spheres, or “beads” (the stationary phase).
 The mass of beads within the column is often referred to as the
column bed.
 The beads act as “traps” or “sieves” and function to filter small
molecules which become temporarily trapped within the pores.
 Larger molecules are “excluded” from the beads .
 Large sample molecules cannot or can only partially penetrate the
pores, whereas smaller molecules can access most or all pores.
 Thus, large molecules elute first, smaller molecules elute later, while
molecules that can access all the pores elute last from the column.
 Particles of different sizes will elute(filter) through a stationary phase
at different rates.
  Two dimensional
On the basis of chromatographic bed shape

i. Thin Layer Chromatography

ii. Paper Chromatography
 Three dimensional
i. Column Chromatography
Thin Layer Chromatography
Definition:
Thin-layer chromatography (TLC) is a
chromatographic technique that is useful for
separating organic compounds. Because of the
simplicity and rapidity of TLC, it is often used
to monitor the progress of organic reactions and
to check the purity of products.
Principle:
Similar to other chromatographic methods TLC is also based
on the principle of separation. The separation depends on the
relative affinity of compounds towards stationary and mobile
phase. The compounds under the influence of mobile phase
(driven by capillary action) travel over the surface of stationary
phase. During this movement the compounds with higher
affinity to stationary phase travel slowly while the others travel
faster. Thus separation of components in the mixture is
achieved.
Once separation occurs individual components are visualized
as spots at respective level of travel on the plate. Their nature
or character are identified by means of suitable detection
techniques.
Paper Chromatography
Definition:
Paper chromatography is an analytical
method that is used to separate coloured chemicals or
substances, especially pigments. This can also be used in
secondary or primary colours in ink experiments. This method
has been largely replaced by thin layer chromatography, but is
still a powerful teaching tool. Double-way paper
chromatography, also called two-dimensional chromatography,
involves using two solvents and rotating the paper 90° in
between. This is useful for separating complex mixtures of
compounds having similar polarity, for example, amino acids. If
a filter paper is used, it should be of a high quality paper. The
mobile phase is developing solutions that can travel up to the
stationary phase carrying the sample along with it.
 Principle:
The principle involved is partition chromatography where
in the substances are distributed or partitioned between to
liquid phases. One phase is the water which is held in
pores of filter paper used and other phase is that of 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.
The principle can also be adsorption chromatography
between solid and liquid phases, where in 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 two liquid
phases.
On the base of physical state of mobile phase
 Liquid Chromatography
 Gas Chromatography
 Super Critical Fluid Chromatography
Liquid Chromatography
Liquid chromatography is a technique used to separate a
sample into its individual parts. This separation occurs based
on the interactions of the sample with the mobile and stationary
phases. Because there are many stationary/mobile phase
combinations that can be employed when separating a mixture,
there are several different types of chromatography that are
classified based on the physical states of those phases. Liquid-
solid column chromatography, the most popular
chromatography technique, features a liquid mobile phase
which slowly filters down through the solid stationary phase,
bringing the separated components with it.
 Liquid Chromatography

Liquid-Liquid Liquid-Solid
Chromatography Chromatography

Stationary
Phase Liquid Solid

Normal Reverse Normal Reverse

Phase Phase Phase Phase
Normal Phase Chromatography:
In normal phase chromatography, the stationary phase is polar,
and so the more polar solutes being separated will adhere more
to the stationary adsorbent phase. When the solvent or gradient
of solvents is passed through the column, the less polar
components will be eluted faster than the more polar ones. The
components can then be collected separately, assuming
adequate separation was achieved, in order of increasing
polarity.
Reverse Phase Chromatography:
In reverse phase chromatography, the polarities of the mobile
and stationary phases are opposite to what they were when
performing normal phase chromatography. Instead of choosing
a non-polar mobile phase solvent, a polar solvent and non-
polar stationary phase will be chosen.
Extraction Chromatography:
An important modification in terms of stationary phase is
introduced by loading the extractant used for solvent extraction
on a hydrophobized inert support and irrigating the support
with aqueous solvent. This is known as extraction
chromatography.
Affinity Chromatography:
Affinity chromatography is a method of
separating biochemical mixtures based on a highly specific
interaction such as that
between antigen and antibody, enzyme and substrate,
or receptor and ligand.
Principle:
The stationary phase is typically a gel matrix, often of agarose; a
linear sugar molecule derived from algae. Usually the starting
point is an undefined heterogeneous group of molecules in
solution, such as a cell lysate, growth medium or blood serum.
The molecule of interest will have a well known and defined
property, and can be exploited during the affinity purification
process. The process itself can be thought of as an entrapment,
with the target molecule becoming trapped on a solid or
stationary phase or medium. The other molecules in the mobile
phase will not become trapped as they do not possess this
property. The stationary phase can then be removed from the
mixture, washed and the target molecule released from the
entrapment in a process known as elution. Possibly the most
common use of affinity chromatography is for the purification
of recombinant proteins.
High Performance Liquid Chromatography
High-performance liquid chromatography (HPLC;
formerly referred to as high-pressure liquid
chromatography), is a technique in analytic chemistry used to
separate the components in a mixture, to identify each
component, and to quantify each component.
It relies on pumps to pass a pressurized
liquid solvent containing the sample mixture through a column
filled with a solid adsorbent material.
Each component in the sample interacts slightly differently
with the adsorbent material, causing different flow rates for the
different components and leading to the separation of the
components as they flow out the column.
Gas Chromatography
Gas chromatography (GC), is a common type
of chromatography used in analytical chemistry for separating and
analyzing compounds that can
be vaporized without decomposition.
Typical uses of GC include testing the purity of a particular
substance, or separating the different components of a mixture
(the relative amounts of such components can also be
determined).
In some situations, GC may help in identifying a compound.
In preparative chromatography, GC can be used to prepare pure
compounds from a mixture.
Two types of gas chromatography are encountered
a. Gas-Solid Chromatography (GSC)
b. Gas-Liquid Chromatography (GLC)
Supercritical Fluid Chromatography
Supercritical Fluid Chromatography (SFC) is a form
of normal phase chromatography, that is used for the analysis
and purification of low to moderate molecular weight,
thermally labile molecules.
It can also be used for the separation of chiral compounds.
 Principles are similar to those of high performance liquid
chromatography (HPLC), however SFC typically
utilizes carbon dioxide as the mobile phase; therefore the entire
chromatographic flow path must be pressurized.
 The supercritical phase represents a state in which liquid and
gas properties converge, supercritical fluid chromatography is
sometimes called "convergence chromatography."
References
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