Chromatography is the science of separation techniques . The technique is used to fractionate mixture of gases, liquids or dissolved solids

1. Chromatographic technique for
life science researchers
Presented by
Dr. B. Victor., Ph.D.,
email : bonfiliusvictor@gmail.com
blog : bonvictor.blogspot.com

2. Presentation outline
Presentation
 Science of chromatography
outline
 Meaning, origin, scope and
definition.
 Principles, Rf value and basic
categories
 Classification of chromatography
 Research applications
 Research choice of
chromatographic methods
 Significance of chromatography.

3. Science of chromatography
What is chromatography?
• Chromatography is the science
of separation techniques .
• The technique is used to
fractionate mixture of gases,
liquids or dissolved solids.

4. Meaning of chromatography
Color writing
• The name chromatography
(Greek: chroma means color and
graphein means writing)literally
means writing in color.
• In other words writing out the
‘signature ‘ of a mixture in color.

5. Origin of chromatographic methods
 The Russian botanist
Mikhail S. Tswett (1872-
1919) found that pigment
composition became
separated when plant
Mikhail S. Tswett pigment (chlorophyll)
(1872-1919) together with petroleum
ether went through calcium
carbonate layer.

6. Scope of chromatography
Statement of Tswett (1906)
• Chromatography is a method in
which the components of a
mixture are separated on an
adsorbent column in a flowing
system.

7. Definitions of chromatography
Definition -1
• Chromatography is a physical
separation technique used to separate
macromolecules based on their size,
shape or charge (Heftmann 1992 ).
Definition -2
• The separation of components of a
mixture based on differential
distribution between a mobile and
stationary phase.

8. Science of chromatography
Chromatography is a
Chromatographic – like
dynamic separation system
separation processes occur
which partitions chemical
in nature –for e.g.
substances between two
migration of water through
phases (Biphasic system)-a
soils results in purification
stationary phase (SP) and a
of water.
mobile phase(MP).
Chromatography is a
science which studies the
Chromatography is a
separation of molecules
flexible yet powerful
based on differences in
analytical procedure.
their structure and / or
composition.

9. Stationary and Mobile phases
in chromatography
Biphasic system
Mobile phase can be
Stationary phase a gas or a liquid which
consists of small solid carries the
particles with micro components of a
porous surface mixture.
The rate of
Stationary phase movement of a given More soluble
The stationary phase
may be packed up in component of a substances travel
may be a solid or
a column or spread mixture depends on more slowly down
liquid supported on a
as a layer or the degree of the column than the
solid or a gel.
distributed as a film. solubility in the less soluble.
solvent system.

10. Basic categories of chromatographic techniques
Based on
Separation Based on
Stationary mobile phase
techniques
phase
Liquid
Liquid-solid
Solid chromatography
Adsorption
Gas
chromatography
Gas-solid
chromatography
Chromatographic
methods
Liquid
Liquid-liquid
Liquid
chromatography
Partition
chromatography Gas
Gas-liquid
chromatography

11. Principle of surface adsorption
Adsorption is the property holding molecules at the surface
of a solid or liquid molecules.
Solute Adsorption coefficient =degree of adsorption
molecules
Molecule
Interaction
specific
Adsorption forces
between
based on Van der vaals forces Electrostatic forces
Solvent Molecular Polar forces chemisorption
molecules structure

12. Surface adsorbents= finely divided porous solids
Choice of Determinants of
adsorbents Adsorption capacity
Silica gel
Alumina Surface area
Powdered sugar Chemical composition
Activated charcoal
Magnesium carbonate
Magnesium silicate Geometrical
arrangements of the
Calcium carbonate groups /atoms
Sodium carbonate

13. Basic characteristics of adsorbents
They are micro
They offer
porous solid They never react
mechanical stability
particles with chemically with
during column
regular or irregular solutes or solvents
packing
surfaces
They retain small
They are
amount soluble
catalytically inactive
components

14. Requirements  Insoluble in mobile phase
of an ideal  Non-reactive to solutes.
adsorbent
 Colorless when separating
colored mixture.
 Choice various particle sizes.

15. kinds of Adsorption chromatography
Adsorption
column
chromatography
Adsorption Thin layer
chromatography chromatography
Ion exchange
chromatography

16. Principle of partition
Partition is the
relative(differential)
solubility of Partition coefficient is
components of a the degree of solubility
mixture in two or more
immiscible solvents.
More soluble
substances travel more
slowly down the column
than the less soluble.

17. Basic characteristics of solvent systems
The composition of the solvent system
should be stable through out the
development.
The developing solvent should move at a
relatively slow rate.
The choice of the solvent should be one in
which the components have small but
definite solubility.

18. Partition solvents
Choice of solvents
 Petroleum ether
 Cyclohexane, carbon
tetrachloride
 Benzene, methylene chloride
 Chloroform
 Ethyl ether, Ethyl acetate
 Pyridine
 Acetone
 Ethanol, methanol
 Water, acetic acid.

19. Partition chromatography
Partition
Column
chromatography
Partition Paper
chromatography chromatography
Thin
layer(Partition)
chromatography

20. Principle of separation
Distribution co-efficient K :
During separation, there is a relationship between
the amount of solute retained by one phase in
relation to another phase.
In partition method the distribution co-efficient is
partition co-efficient i.e. solubility of solute in the
stationary and mobile phases.
In adsorption method , the distribution co-efficient
is adsorption co-efficient i.e. the amount solute
adsorbed by the stationary and mobile phases.

21. Principle of separation
Solvent flow
A B A B A B
AB
Stationary
organic phase Wall of
column
Sample Component A Component B
Mixture

22. Rf value : relative front
The relation of the distance travelled by
compound to that of the solvent front is
called Rf value.
Rf value = Distance travelled by the solute
------------------------------------------
Distance travelled by the solvent
Parameter influencing Rf value
Temperature, Solvent system
Direction of flow, Type of paper.

23. Kinds of chromatography
Gas-solid
Chromatography
Gas mobile GSC
phase Gas-solid
Chromatography
GLC
Column chromatography Liquid-solid
Chromatography
LSC
Liquid-liquid
Liquid mobile
Chromatography
phase
LLC
Chromatographic
techniques
Bonded Phase
Chromatography
Paper
chromatography
Liquid mobile
Planar chromatography
phase
Thin layer
chromatography

24. Chromatographic techniques based
solvent system
Uni- Two
dimensional dimensional
Single Solvent Two
System Solvent systems
One directional
Two directions
solvent flow

25. Modifications of chromatographic methods -1
Multiple development Continuous development
 The paper is developed  The chromatogram is
with the same solvent developed in the same
system several times in solvent system in the
the same direction. same direction
continuously for a long
time.

26. Modifications of chromatographic methods -2
Bidirectional Reverse phase
chromatography chromatography
 The chromatogram is  For the separation of
first developed in one hydrophobic
solvent system in one compounds like fatty
direction. acids, the filter paper is
 It is again developed in treated with lipophilic
a second solvent compounds (silicon
system in direction greese, kerosene oil),
perpendicular to first while the mobile phase
run. is hydrophilic in
nature.

27. Modifications of chromatographic methods-3
One way or Linear flow of solvent
• Column, strips, sheets.
• Thin layer of sorbent
Two –way or transverse solvent flow
• sheets
• Thin layer
Radial or horizontal Solvent flow
• Circular paper discs
• Paper Sheet

28. Types of chromatographic methods
Based on Based on
solvent
quantity direction
Micro-scale
Up-ward
Laboratory assays
ascending
Paper, TLC
Medium scale
Down ward
Analytical assays
descending
Column flow
Large scale Horizontal
Commercial Transverse
GLC, GSC radial

29. Chromatographic development :
principles of separation-1
Elution development Gradient elution
 The components of the  A gradual change in
mixture are separated composition of the eluting
solvent is used to achieve
into zones by the separation of compounds
passage of one or more of widely varying affinities
solvents through the for the stationary phase.
column.  The solvent composition
 This technique is most gradient may be linear with
widely used in GC, increasing or decreasing
concentration, pH, polarity
GLC, LLC and LSC. or ionic strength.

30. Chromatographic development :
principles of separation-2
Frontal analysis Displacement analysis
 No solvent is used for  The components in the
mixture are adsorbed on
irrigation. the column.
 The solution itself is  The irrigation of the
added continuously. column is carried out with
the solution of another
substance, having a higher
preferential adsorption on
the column than that of the
components of the mixture
sought.

31. Research applications of
chromatographic methods
Analytical applications Preparative applications
 To determine the  To purify and collect
chemical composition one or more
of a biological sample. components of a
biological sample.

32. Research choice of chromatographic methods
Substances
Of similar chemical type
• Partition chromatography
Substances of different
chemical type • Adsorption chromatography
Gases and volatile
substances
• Gas chromatography
Ionic and inorganic • Ion exchange chromatography
substances • Column or paper or thin layer
Biological materials and
Compounds of relative • Gel chromatography
High molecular mass

33. Protocol of chromatography methods
Column chromatography Paper chromatography
 A mixture of components  The paper adsorbs
dissolved in a solvent is water from the
poured over a column of
solid adsorbent . atmosphere of the
 The column is eluted developing
with the same or a chromatogram.
different solvent.  The water is the
 The stationary phase is stationary phase.
solid.
 The eluting solvent is
 The mobile phase (the
eluent) is liquid. the mobile phase.

34. Significance of chromatographic methods
They serve to resolve Very small quantities
and identify the of substances could
The equipment is very
separated be analyzed
simple except HPLC
components of a qualitatively and
mixture. quantitatively.
No special skill is
The results are
required for
remarkably
performing the
reproducible.
method

35.  Dr.B.Victor is a highly experienced professor,
recently retired from the reputed educational
institution- St. Xavier’ s College, Palayamkottai,
India-627001.
 He was the dean of sciences, IQAC coordinator and
assistant controller of examinations.
 He has more than 32 years of teaching and research
experience
 He has taught a diversity of courses and guided 12
Ph.D scholars.
 Send your comments to :
bonfiliusvictor@gmail.com