BASIC CONCEPT OF CHROMATOGRAPHY
INTRODUCTION TO CHROMATOGRAPHY
• Defination - chromatography is a separation technique based on the different
interactions of compound with two phases , a mobile phase and a stationary phase as
the compound travel through the a supporting medium.
• COMPONENTS:
Mobile phase- a solvent that flows through the supporting medium.
Stationary phase- a layer or coating on the supporting medium that interacts
with the analytes.
Supporting medium - a solid surface on which the stationary phase is bound
or coated.
1. chromatography is a technique used to separate and identify
the components of the mixture.
2. Works by allowing the molecule present in the mixture to
3. distribute themselves b/w a stationary and a mobile medium.
4. Chroma means “color” and graphien means “to write”.
Colour bands –separation of individual compounds.
HISTORY
1903 Tswett –plant pigment separated on chalk
columns .
1931 Lederer & Kuhn – LC on caretonoids .
1938 TLC and ion exchange
1950 reverse phase LC
1954 martin & synge ( nobel prize)
1959 gel permeation
1965 instrumental LC ( waters)
CHROMATOGRAPHY TERMS
1. Chromatograph – equipment that enables a sophisticated
separation of Eg: gas chromatography or liquid
chromatography
2. ELUENT – fluid entering column /solvent that carries the analyte .
3. Eluate – mobile phase leaving the column .
4. Stationary phase – immobilized phase
. Immobilized on the support partical or on the inner wall of the column tubing.
eg. Silica layer – thin layer chromatography
CHROMATOGRAPHY TERMS
Mobile phase- moves in a definite direction . Liquid (LC), GAS (GC). The mobile phase moves
through the chromatography column (stationary phase ) where the sample interacts with the
stationary phase and is separated.
Retention Time- time taken for a particular analyte to pass through the the column inlet to the
detector under set condition.
Sample- substance analyzed in chromatography .
Solvent- any substance capable of solubilizing another substance .
PRINCIPLE OF CHROMATOGRAPHY
Physical method of separation that distribute components to separate that
distribute to separate b/w two phases moves in a direction.
substances are separated based on their differential distribution b/w two
phases.
substances will move with the mobile phase at different rate depending
upon their partition or distribution co-efficient
PRINCIPLE OF CHROMATOGRAPHY
.
T H E ANALYTES INTERACTING MOST
STRONGLY
with the stationary phase will take
longer to pass through the system
than those with weaker interaction.
These interaction are usually
che mical in nature, but in some ca ses
physical interaction can also be used.
This Photo by Unknown Author is licensed under CC BY-SA-NC
This Photo by Unknown Author is licensed under CC BY-SA
CHROMATOGRAM
. Visual output of the
chromatograph
. separation –different peaks
or patterns on the This Photo by kn Author is licen nder CC BY-NC
chromatogram correspond
to different components of
the to the separated mixture
CLASSIFICATION OF CHROMATOGRAPHY :
H
CLASSIFICATION OF CHROMATOGRAPHY
• There are two classification
schemes:
Mobile phase
Attractive forces
Mobile phase:
gas (GC)
water (LC)
organic solvent
supercritical fluid (SCFC)
CLASSIFICATION OF CHROMATOGRAPHY
Gas Liquid
chromatography chromatography
attractive forces
On the basis of
adsorption
Gas –solid Column (gravity flow) Ion exchange
Partition
High performance
Size exclusion
Gas liquid (pressure flow )
.Thin layer
(adsorption )
CLASSIFICATION OF CHROMATOGRAPHY
Techniques by chromatographic bed shape
Column chromatography
Planar chromatography Paper chromatography Thin
layer chromatography
Techniques by physical state of mobile phase Gas chromatography,
Liquid chromatography
Affinity chromatography Supercritical fluid chromatography
COLUMN CHROMATOGRAPHY
Adsorption /retention of substances on
stationary phase
Separation of absorbed substance using
mobile phase
Recovery of individual components by
continuous flow of mobile phase .
This Photo known Aut censed unde CC BY-SA-NC
Quantitative and qualitative analysis of solute
and the components which are recovered.
PLANAR CHRAMTOGRAPHY
Separation techniques- stationary phase is present as
or on a plane .
paper-paper Chromatography
Layer of solid particles spread on a support such as
a glass plate –thin layer chromatography
Different compound in the sample mixture travel
different distance according to how strongly they
interact with the stationary phase as compared to
the mobile phase
retention factor (RF).
This Photo by Unknown Author is licensed under CC BY-SA
PAPER CHROMATOGRAPHY
Principle
This paper is made up of cellulose ,a polar
substance ,and the compounds within a
mixture travel farther if they are non polar
More polar substances bond with the
cellulose paper more quickly ,and therefore
do not travel as far .
Retention factor (rf): distance
travelled by a solute /distance travelled by
the solvent
Rf= zero ,-solute remains in the stationary
phase and thus it is immobile.
RF=1 , -solute has no affinity for the
stationary phase and travels with the
solvent front.
This Photo by Unknown Author is licensed under CC BY-SA-NC
THIN LAYER CHROMATOGRAPHY
Widely employed laboratory techniques
Stationary phase –adsorbent –silica gel,
alumina, cellulose
Widely used in pharmaceutical & food stuff industry
Advantages
• simple ,rapid and cheap .
• Faster runs
• Better separation
• Choice b/w different
adsorbents
• Better resolution
• Allow for quantification
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GAS CHROMATOGRAPHY
Gas liquid chromatography (GLC).
Mobile phase –gas(helium) carrier gas pressure =4kg/cm2
Stationary phase –column ,which is tightly packed or
“capillary”.
The stationary phase adhered to the inside of a small
diameter glass tube(a capillary column) or a solid matrix
inside a larger metal tube (a packed column).
Partition coefficient of volatile analyte b/w a solid
stationary
phase (silicone) and a mobile gas.
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GAS CHROMATOGRAPHY
ADVANTAGES:
High sensitivity
High resolution
High speed
High accuracy
Highly quantitative
APPRATUS:
Gas chromatograph ,GC analyzer , normal
syringes and one micro syringe ,beakers ,
sample
bottles and electronic weight.
CHEMICALS:
Methanol ,isopropyl alcohol and water .
SAMPLE :
Gases ,liquid ,solids .
M.wt: 2-800 This Photo by Unknown Author is licensed under CC BY-SA
volatile
APPLICATION OF GAS CHROMATOGRAPHY
Quantitative &qualitative analysis of low polarity compounds .
Analytical chemistry ,biochemistry ,petrochemical , environmental monitoring .
Measures picomoles of a substance in a 1 ml liquid samples or part per billion concentration in gaseous
samples .
Measuring toxic substances in soil ,air or water.
Environmental monitoring
Criminal forensics
Law enforcement
Forensic toxicology
Sport anti doping analysis
Security
Food ,beverages and perfume
Medicine
LIQUID CHROMATOGRAPHY
Mobile phase –liquid
Column or a plane
Very small packing and a relatively high pressure –
high performance liquid chromatography (HPLC)
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SOLVENT TYPE USED IN LIQUID CHROMATOGRAPHY
ORGANIC
Methanol
Acetonitrile
THF
Methylene chloride
Hexane
AQUEOUS
Water
SOLVENT MIXTURES
Chcl3/ETOH
MeOH/CAN /h2o
Hexane /IPA
This Photo by Unknown Author is licensed under CC BY-SA
ADSORPTION CHROMATOGRAPHY
Adsorption chromatography is probably one of
the oldest types of chromatography around
.
It utilizes a mobile liquid or gaseous phase
that is adsorbed onto the surface of a
stationary solid phase .
The equilibration b/w the mobile and stationary
This Photo by Unknown Aut CC BY-SA
phase accounts for the separation of different
solutes .
ION EXCHANGE CHROMATOGRAPHY
In this type of chromatography ,the use
of a resin (the stationary) is used to
covalently attach anions or cations
onto it.
Solute ions of the opposite charge in
the mobile liquid phase are attracted to
the resin by electrostatic forces .
This Photo by Unknown Author is licensed under CC BY-SA-NC
PARTITION CHROMATOGRAPHY
THIS form of chromatography is based on a thin film formed on the surface of a solid support by a
liquid stationary phase .solute equilibrates b/w the mobile phase and a stationary liquid.
This Photo CC BY
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