chromatography by pillai aswathy viswanath

1. PRINCIPLES AND APPLICATION OF
CHROMATOGRAPHY
By,
Pillai Aswathy viswanath
PG 2 Botany
St. Thomas college
kozhencherry

2. INTRODUCTION
 The word chromatography means "color
writing“
 It is an important technique employed for
the separation , isolation and identification
of the components of a mixture
 Chromatography is a method used by
scientists for separating organic and
inorganic compounds.
 This method is particularly useful when the
components in the mixture have almost the
same physical and chemical properties

3.  Some materials appear homogenous,
but are actually a combination of
substances. For example, green plants
contain a mixture of different
pigments.
 While studying the coloring materials
in plant life, a Russian botanist
invented chromatography in 1903. His
name was M.S. Tswett.

4.  He continued to work with
chromatography in the first decade of
the 20th century, primarily for the
separation of plant pigments such as
chlorophyll, carotenes, and xanthophylls.
 Since these components have different
colors (green, orange, and
yellow,respectively) they gave the
technique its name.

5. PRINCIPLES
 In all chromatography there is a mobile
phase and a stationary phase.
 The stationary phase is the phase that
doesn't move and the mobile phase is
the phase that does move.
 The mobile phase moves through the
stationary phase picking up the
compounds to be tested.

6.  As the mobile phase continues to travel
through the stationary phase it takes
the compounds with it.
 At different points in the stationary
phase the different components of the
compound are going to be absorbed and
are going to stop moving with the mobile
phase.

7.  This is how the results from the point
at which the different components of
the compound stop moving and separate
from the other components.
 The interaction between the mobile
phase and the stationary phase results
in the separation of the compound from
the mixture.

8. There are four main types of
chromatography. These are:-
 Paper Chromatography.
 Thin-Layer Chromatography
 Gas Chromatography,
 Liquid Chromatography,

9. PAPER CHROMATOGRAPHY
 Paper chromatography is considered
to be the simplest and most widely
used
 Paper chromatography is the method
in which the analysis of an unknown
substance is mainly done by the flow
of solvent on specially designed filter
paper

10.  Paper chromatography is a technique
that involves placing a small dot or line
of sample solution onto a strip of
chromatography paper.
 The paper is placed in a jar containing a
shallow layer of solvent and sealed.
 As the solvent rises through the paper,
it meets the sample mixture, which
starts to travel up the paper with the
solvent.

11.  Capillary action is used to pull the
solvents up through the paper and
separate the solutes.
 A strip of paper as the stationary phase.

12. APPLICATION
 Separating amino acids and anions,
 RNA fingerprinting,
 Separating and testing histamines,
 Antibiotics

13. THIN-LAYER CHROMATOGRAPHY
 Thin layer chromatography (TLC) is a
widely employed laboratory technique
and is similar to paper chromatography.
 However, instead of using a stationary
phase of paper, it involves a stationary
phase of a thin layer of adsorbent like
silica gel, alumina, or cellulose .
 Compared to paper, it has the
advantage of faster runs, better
separations, and the choice between
different adsorbents.

15. APPLICATION
 Thin-layer Chromatography uses an
absorbent material on flat glass or
plastic plates.
 This is a simple and rapid method to
check the purity of an organic
compound.
 It is used to detect pesticide or
insecticide residues in food.
 Thin-layer chromatography is also used
in forensics to analyze the dye
composition of fibers.

16. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC )
 Liquid chromatography is variously known
as high pressure , high speed or high
performance liquid chromatography
 Liquid chromatography is a separation
technique in which the mobile phase is a
liquid.
 Liquid chromatography can be carried
out either in a column or a plane.

17.  In HPLC the sample is forced by a
liquid at high pressure (the mobile
phase) through a column that is
packed with a stationary phase
composed of irregularly or spherically
shaped particles.
 The interaction between the mobile
and the stationary phase leads to the
separation of the mixture.

18.  Present day liquid
chromatography that
generally utilizes very
small packing particles
and a relatively high
pressure is referred to
as high performance
liquid chromatography
(HPLC).

19.  The chromatographic techniques are
slow & time consuming, hence the
separation can be greatly improved by
using high pressure in the range of
5000-10000 psi(pounds per square
inch),hence this technique is also
referred to as high pressure liquid
chromatography.

20. APPLICATION
 It is used in the world to test water
samples to look for pollution in lakes and
rivers.
 It is used to analyze metal ions and
organic compounds in solutions.
 Liquid chromatography uses liquids which
may incorporate hydrophilic,insoluble
molecules.

21. GAS CHROMATOGRAPHY
 One of the newest and most versatile
technique for analyzing complex mixture
 Gas chromatography also sometimes
known as Gas-Liquid chromatography
 It is a separation technique in which the
mobile phase is a gas.
 Stationary phase is an inert solid
material impregnated with a non-volatile
liquid.

22.  Here the mixture is separated in to
constituents by a moving gas phase
passing over a stationary adsorbent
 The separation of the mixture is based
on the partition of the components
between the mobile phase(gas) and
stationary phase (liq.) hence the name
Gas-liquid Chromatography.

23.  It is the method of
choice for the separation
of volatile substances or
the volatile derivatives
of certain non- volatile
substances.
 In gas chromatography
helium is used to move a
gaseous mixture through
a column of absorbent
material.

24. APPLICATION
 It is used in airports to detect bombs
and is used is forensics in many
different ways.
 It is used to analyze fibers on a persons
body and also analyze blood found at a
crime scene.
 Largely used for the chromatographic
separation of aromatic isomers like
xylenes,toluidines,cresols and
dichlorobenzene