CHROMATOGRAPHY:
Principle and applications
A level
2021-22
BIOLOGY
LEARNING OBJECTIVE
• To know about the chromatography.
• To know about principle, Rf value
• Differentiate types of chromatography.
INTRODUCTIO
N
Chromatography is a physical process where the
Stationary
components (solutes) Phas
e
of a sample mixture
are separated as a
result of their differential
distribution between
stationary and mobile
phases.
Greek chroma meaning
‘color’ and graphein meaning ‘writing’
HISTORY
Tswet, Russian botanist
(referred to as Father of
chromatography) is credited for
the development of
chromatography.
Principle -
Chromatography is usually based on principle of partition of
solute between two phases. It usually consists of a Mobile
Phase and a Stationary Phase.
The Mobile Phase usually refers to the mixture of the
substances to be separated dissolved in a liquid or a gas .
The Stationary Phase is a porous solid matrix through which
the sample contained in the mobile phase percolates.
CHROMATOGRA
M
A graphical presentation of detector response,
concentration of analyte in the
effluent, or other quantity used
as a measure of effluent
concentration.
The retention time or volume is
when a solute exits the injector
and passes through the
column and the detector .
Data represented by the chromatogram are used to help
identify and quantify the solute(s).
Because eluting solutes are
displayed graphically as a
series of peaks, they are
frequently referred to as
chromatographic peaks.
These Peaks are described in
terms of peak (1) width,
(2)height, (3)area
CLASSIFICATION
Chromatographic methods can be classified in three different
ways :-
a) Based on shape of chromatographic beds .e.g.- Planar and
column Chromatography
b) Based on the physical state of mobile and stationary phase.
e.g- Gas and liquid chromatography
c) Based on mechanism of separation. e.g.-Ion-exchange
chromatography, partition , affinity and adsorption
chromatography
Based on shape of
chromatographic beds
Chromatography
Plana Colum
r n
Thin Layer
GAS Liquid
Pape
r (TLC)
(GC (LC)
)
Planar Chromatography
In Planar Chromatography stationary phase is
present on a plane.
The Plane can be apaper impregnated by a substance
acting as a stationary phase- Paper Chromatography
OR a Thin layer of a substance acting as a
stationary phase spread on a glass, metal or plastic
plate- Thin Layer Chromatography.
Planar chromatography is also termed as Open Bed
Chromatography.
Procedur
e
A small spot of sample is applied to a strip of chromatography paper about
two centimeters away from the
base of the plate.
This sample is absorbed onto the paper
and may form interactions with it.
The paper is then dipped into a solvent,
such as ethanol or water, taking care
that the spot is above the surface of the
solvent , and placed in a sealed container
.
Significance of Paper
Chromatography
It is very easy, simple , rapid and highly efficient method of
separation.
Can be applied in even in micrograms quantities of the
sample.
Can also be used for the separation of a wide variety of material
like amino acids , oligosaccharides, glycosides, purines and
pyrimidines, steroids, vitamins and alkaloids like penicillin ,
tetracyclin and streptomycin.
Thin Layer Chromatography (TLC)
• Stationary Phase consists of a thin layer
of adsorbent material, usually silica gel ,
aluminium oxide, or cellulose immobilized
onto a flat carrier sheet.
n
• A Liqiud Phase consisting of the solutio
to be separated which is dissolved in
• an appropriate solvent and is drawn up the plate via
capillary action , separating the solution based on
the polarity of the compound.
Steps of
TLC
Significance
Its wide range uses include -
Determination of the pigments a plant contains.
Detection of pesticides or insecticides in food .
Identifying compounds present in a given substance.
Monitoring organic reaction.
R value
F
The rate of migration of the various
substances being separated are governed by
their relative solubilities in the polar
stationary phase and non polar mobile phase.
The migration rate of a substances usually
expressed as Rf (relative front).
Rf = Distance travelled by the substance
Distance travelled by the solvent front
Column Chromatography
The Stationary bed is within the tube.
In column Chromatography the
stationary Phase may be pure silica
or polymer, or may be coated onto ,
chemically bonded to, support
particles.
Depending on whether mobile phase is a gas or a liquid it is divided
into- gas Chromatography or liquid Chromatography.
When the Stationary phase in LC consists of small-diameter particles, the
technique is High Performance Liquid Chromatography (HPLC).
Gas
Chromatography
Gas mobile phase is used to pass a mixture of volatile
solutes through a column containing the stationary
phase.
The mobile phase often referred to as the carrier gas , is
typically an inert gas such as nitrogen, helium ,or argon.
Solute separation is based on the relative differences in
the solutes vapor pressures and interactions with the
stationary phase.
Thus more volatile solute elutes from the column
A solute that selectively interacts with the stationary
phase elutes from the column after with lesser
degree of interaction.
The column effluent carries separated solutes to the
detector in order of their elution.
Solutes are identified qualitatively by their retention
times.
Peak size is proportional to the amount of solute
detected and is used to quantify it.
Liquid Chromatography
Separation by LC is based on the distribution of the solutes between a liquid
mobile phase and a stationary phase.
When particles of small diameter are used as stationary phase support, the
technique is HPLC.
Most widely used form of LC.
Instrumentation
A basic Liquid chromatograph consists of following elements :-
A solvent reservoir to hold the mobile phase through the system.
An injector to introduce sample into the column.
A chromatographic column to separate the solutes.
Detector to detect the separated analytes as they elute from the column.
A computer that processes the system and processes data.
Application
s
Pharmaceutical- Tablet dissolution of pharmaceutical dosages
Shelf life determination of pharmaceutical products
Identification of counterfeit drug products
Pharmaceutical quality control
Forensics- On site identification and quantification of the drug Ecstasy.
Identification of anabolic steroids in serum, urine, sweat and hair
Forensic determination of textile dyes.
Simultaneous quantification of psycotherapeutic drugs in human plasma
Clinical-
Analysis of antibiotics.
Detection of endogenous neuropeptides in brain extracellular
fluids.
Food and Flavour-
Ensuring soft drink consistency and quality
Analysis of vicinal diketones in beer.
Sugar analysis in fruit juices.
Trace analysis of military high explosives in agricultural crops.
Based on Separation
Mechanisms
Chromatographic separations are classified by the
chemical or physical mechanisms used to separate
solutes.
These include-
1. Ion- exchange
2. Partition
3. Adsorption
4. Size exclusion
5. Affinity mechanisms
LEARNING OBJECTIVE
• To know about the chromatography.
• To know about principle, Rf value
• Differentiate types of chromatography.
LEARNING OBJECTIVE
• To know about the chromatography.
• To know about principle, Rf value
• Differentiate types of chromatography.
