2. • Chromatography-
• Separation technique involving mass transfer
between stationary and mobile phase.
• In Greek chromo meaning “color”, Graphic
meaning “writing”.
• Chromatography is used in industrial processes to
purify chemicals, test trace quantities of
substances, separate chiral compounds and quality
control test products. Chromatography is the
physical process of separating or analyzing
complex mixtures.
3. History
• Chromatography is a powerful separation method that
finds applications in all branches of science,
Chromatography was invented and named by the
Russian botanist Mikhail Tswett shortly after the turn
of the last Century.
• He employed the technique to separate various plant
pigments such as chlorophylls and xanthophylls by
passing solutions of these compounds through a glass
column packed with finely divided calcium carbonate,
The separated species appeared as colored bands on
the column, which accounts for the name he chose for
the method (Greek chroma meaning "color”and graphe
in meaning "writing")
4. Chromatography Definition
• Chromatography is an important biophysical technique
that enables the separation, identification, and
purification of the components of a mixture for
qualitative and quantitative analysis.
• A wide range of chromatographic procedures makes use
of differences in size, binding affinities, charge, and other
properties to separate materials.
• It is a powerful separation tool that is used in all
branches of science and is often the only means of
separating components from complex mixtures.
• Chromatography is a very useful technique as it allows
the separation of components of a mixture on the basis
of their nature, structure, size, and other properties.
5. Principle of Chromatography
• Chromatography is a separation method where the
analyte is contained within a liquid or gaseous
mobile phase, which is pumped through a stationary
phase.
• Usually, one phase is hydrophilic and the other
lipophilic. The components of the analyte interact
differently with these two phases.
• Depending on the polarity they spend more or less
time interacting with the stationary phase and are
thus retarded to a greater or lesser extent.
• This leads to the separation of the different
components present in the sample.
6. • Chromatography, in general, is based on the
principle that components of a mixture are
separated when the mixture added to a mobile
phase is moved through a stationary phase
(which mostly is a solid surface), resulting in some
components of the mixture being attached to the
stationary phase. At the same time, the rest is
passed along with the mobile phase.
• Thus, there are two essential components of all
chromatography techniques.
7. What is a stationary phase?
• The stationary phase in chromatography is the
phase that is either a solid or liquid particle
attached to a glass or a metal surface on
which the components of the mixture to be
separated is absorbed selectively.
• The term stationary refers to the fact that this
phase remains stationary while the other
phase moves.
• Most substances used as stationary phases are
porous, thus allowing the attachment of
components during chromatography.
8. • The stationary phase to be selected for a
chromatographic process depends on the
nature of the components to be separated
and the type of chromatography.
• Depending on the type of chromatography gel
beads, thin uniform paper, silica, glass, some
gases, or even liquid components are used as
a stationary phase.
9. What is the Rf value
• The Rf value is defined as the ratio of the
distance moved by the solvent (i.e. the
colouring or pigment being tested) and the
distance moved by the solvent (known as the
Solvent front) along the paper, where both
distances are measured from the common
origin or application baseline.
10.
11. What is the mobile phase?
• The mobile phase in chromatography is the
phase that is either liquid or gas that is passed
through a chromatographic system where the
components of the mixture are separated at
different raters by adsorbing them to the
stationary phase.
12. • The mobile phase is the solvent that carries the
mixture as it moves down the stationary phase.
• The term mobile indicates that the phase is moving
down the chromatographic system whereas the
other phase remains stationary.
• Substances used as mobile phases are selected for a
chromatographic process depending on the nature
of the components to be separated and the type of
chromatography.
• Alcohol, water, acetic acid, acetone, or some gases
are the commonly used mobile phase in different
chromatographic techniques.
13. What is the main purpose of
chromatography?
• Preparative chromatography is intended to
isolate the components of a mixture for
subsequent use and is, therefore, a method of
purification.
• Analytical chromatography is usually done
with smaller amounts of material and is
intended to determine the presence of the
relative proportions of analytes in a mixture.
14. Types
• Paper chromatography
• Thin layer chromatography
• Affinity chromatography
• Ion exchange chromatography
• Column chromatography
• Size exclusion / gel filtration chromatography
• Liquid chromatography
• HPLC
• Gas chromatography
15.
16. The applications of chromatography have grown
explosively in the last half century, due not
only to the development of several new types
of chromatographic techniques but also to the
growing need by scientists or better methods
for characterizing complex mixtures.
17. The tremendous impact of these methods on
science is attested by the 1952 Nobel Prize in
Chemistry hat was awarded to A. J P,Martin and
R. LM, Synge or their discoveries in the field,
Many of the Nobel Prizes awarded since that
time have been based on work in which
chromatography played a vital role.
18. Description
• Chromatography encompasses a diverse and
important group of methods that allow the
separation, identification, and determination of
closely related components of complex mixtures;
many of these separations are impossible by
other means.
• ' In all chromatographic separations the sample is
dissolved in a mobile phase, which may be a gas,
a liquid, or a supercritical fluid.
19. This mobile phase is then forced through an
immiscible stationary phase, which is fixed in
place in a column or on a solid surface. The two
phases are chosen so that the components of the
sample distribute themselves between the
mobile and stationary phases to varying degrees
20. • Those components strongly retained by the
stationary phase move only slowly with the
flow of mobile phase. In contrast, components
that are weakly held by the stationary phase
travel rapidly.
• As a consequence of these differences in
migration rates, sample components separate
into discrete bands, or zones, that can be
analyzed qualitatively and quantitatively.
21. Classification of
Chromatographic Method
Chromatographic methods can be categorized in two ways. The first
classification is based on the physical means by which the
stationary and mobile phases are brought into contact.
In column chromatography, the stationary phase is held in a narrow
tube through which the mobile phase is forced under pressure.
In planar chromatography, the stationary phase is supported on a
flat plate or in the interstices of paper; here, the mobile phase
moves through the stationary phase by capillary action or under
the influence of gravity.
22. The discussion in this and the next three chapters
focuses on column chromatography.
It is important to point out here, however, that the
equilibria on which the two types of chromatography
are based are identical and that the theory developed
for column chromatography is readily adapted to
planar chromatography.
23. • A more fundamental classification of
chromatographic methods is one based on the
types of mobile and stationary phases and the
kinds of equilibria involved in the transfer of
solutes between phases.
• A more fundamental classification of
chromatogra-phic methods is one based on the
types of mobile and stationary phases and the
kinds of equilibria involved in the transfer of
solutes between phases.
24. Three general categories of chromatography: gas
chromatography (GC), liquid chromatography (LC), and
supercritical fluid chromatography (SFC).
As the names imply, the mobile phases in the three
techniques are gases, liquids, and supercritical fluids,
respectively.
Several specific chromatographic methods fall into each of
the first two general categories.
Note that only LC can be performed either in columns or
on planar surfaces; GC and SFC, on the other hand, are
allowed only to column procedures so that the column
walls contain the mobile phase.
25.
26. What are the advantages of chromatography?
• Using chromatography, precise separation,
analysis, and purification are possible.
• It works on a variety of samples including
drugs, food particles, plastics, pesticides,
samples of air and water, and extracts of
tissue.
