Chromatography is a technique used to separate mixtures by exploiting differences in how components interact with a stationary and mobile phase. There are four main types: paper chromatography uses paper as the stationary phase; thin-layer chromatography uses silica gel or other adsorbents; gas chromatography uses an inert solid and gas as phases to separate volatile compounds; and high performance liquid chromatography forces liquid samples through columns under high pressure for improved separation. Chromatography has many applications across fields like analyzing plant pigments, detecting pesticides, identifying pollutants, and aiding forensic investigations.

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