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Chromatography

Chromatography is a physical process used to separate components of a mixture based on their distribution between stationary and mobile phases. It can be classified by the shape of chromatographic beds, the physical state of phases, and mechanisms of separation, including methods like paper chromatography, thin layer chromatography, gas chromatography, and liquid chromatography. Applications of chromatography span various fields such as pharmaceuticals, forensics, clinical analysis, and food quality control.

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21 views29 pages

Chromatography

Chromatography is a physical process used to separate components of a mixture based on their distribution between stationary and mobile phases. It can be classified by the shape of chromatographic beds, the physical state of phases, and mechanisms of separation, including methods like paper chromatography, thin layer chromatography, gas chromatography, and liquid chromatography. Applications of chromatography span various fields such as pharmaceuticals, forensics, clinical analysis, and food quality control.

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abilasha rajen
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© © All Rights Reserved
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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.

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