• 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

1. Presented By: Jagdish Chandra Jat
Sangam University, Bhilwara

2. 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 redistributes 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.

3. IllustrationofChromatography
Separation
Mixture
Mobile Phase
Components

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

5. ExamplesofChromatography
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.
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.

6. 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.

7. • 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.

8. 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.
• 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-

9. 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.

10. • 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
canbe named as the century of chromatography.

11. Classification of Chromatography
On the basis of interaction of solute
to the stationaryphase
On the basis of chromatographic bed
shape
On the basis of physical state
of mobile phase
Adsorption
Chromatography
Ion Exchange
Chromatography
Two
Dimensional
Three
Dimensional
Super Critical
Fluid
Chromatography
Partition
Chromatography
Size Exclusion
Chromatography
Column
Chromatography
Gas
Chromatography
Thin Layer
Chromatography
Paper
Chromatography
Liquid
Chromatography

12. On the basis of interactionof Solute to the
stationary phase
➢ Adsorption Chromatography
➢ Partition Chromatography
➢ Ion Exchange Chromatography
➢ Size Exclusion Chromatography

13. AdsorptionChromatography
• 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.

14. 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.

15. Gas Solid
Chromatography
Thin Layer
Chromatography
Column
Chromatography
Adsorption Chromatography
Types:

16. PartitionChromatography
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.

17. 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
appears separated because of differences in their partition
coefficient.

18. Gas-liquid
Chromatography
Liquid-liquid
Chromatography
Partition Chromatography
Types:

19. IonExchangeChromatography
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.

21. 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.

22. Types:
Ion Exchange
Chromatography
Cation Exchange
Chromatography
Solute cations are attached to the
negatively charged sites covalently
bond to the stationary phase
Solute anions are attached to the
positively charged sites covalently
bond to the stationary phase
Anion Exchange
Chromatography

23. SizeExclusionChromatography
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.

24. Size Exclusion Chromatography

25. 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.

26. Onthebasisofchromatographic
bed shape
➢ Two dimensional
i. Thin Layer Chromatography
ii. Paper Chromatography
➢ Three dimensional
i. Column Chromatography

27. ThinLayerChromatography
• 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.

28. • 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 is identified by means of suitable detection
techniques.

29. PaperChromatography
• 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.

31. • 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.

32. On thebaseof physical state ofmobile
phase
➢ Liquid Chromatography
➢ Gas Chromatography
➢ Super Critical Fluid Chromatography

33. LiquidChromatography
• 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.

34. Liquid-Solid
Chromatography
Liquid-Liquid
Chromatography
Liquid Chromatography
SolidLiquid
Normal Phase Reverse Phase Normal Phase Reverse Phase
Stationary
Phase

35. 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.

36. 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.

37. 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.

38. 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.

39. 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.

40. 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.

42. GasChromatography
• 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)

44. SupercriticalFluidChromatography
• 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."

47. References
• http://www.waters.com/webassets/cms/category/media/other_images/primer_T_Ion_Exchange
_CHromatography.jpg
• http://xray.bmc.uu.se/Courses/MPC/students_files/ION_EXCHANGE_CHROMATOGRAPH
Y.pptx
• http://www.thefreedictionary.com/chromatography
• http://www.britannica.com/EBchecked/topic/115917/chromatography/80518/Liquid-
chromatography
• http://www.ltt.com.au/simulab/5/Laboratory/StudyNotes/snClassifChromatoMeth.htm
• http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/be_types.htm
• http://en.wikipedia.org/wiki/Paper_chromatography
• http://en.wikipedia.org/wiki/Aqueous_normal-phase_chromatography
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