Chromatography is a technique used to separate mixtures by distributing components between a stationary and mobile phase. It works on the principle that different compounds interact differently with the phases and therefore move through the system at different rates. There are various types of chromatography classified by mobile phase (gas or liquid) or interaction forces (adsorption, partition, ion exchange). Key components are the mobile phase, stationary phase, and supporting medium. Chromatography is widely used in fields like analytical chemistry, biochemistry, environmental analysis and forensic science.

1. BASIC CONCEPT OF
CHROMATOGRAPHY
Name –Prashant Sharma
Department of biotechnology
seminar

2. 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 .

3. 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
distribute themselves b/w a stationary and a mobile medium .
3. Chroma means “color” and graphien means “to write”.
4.Colour bands –separation of individual compounds.

4. 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)

5. 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

6. • To be continued chromatographic 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 takes for a particular analyte to pass
through the system (from the column inlet to the detector) under
set condition .
• SAMPLE- substance analyzed in chromatography .
• SOLVENT- any substance capable of solubilizing another substance .

7. 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

8. PRINCIPLE OF CHROMATOGRAPHY

9. .
The 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
chemical in nature, but in some cases
physical interaction can also be used.
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10. CHROMATOGRAM
. Visual output of the
chromatograph
. separation –different peaks
or patterns on the
chromatogram correspond
to different components of
the to the separated mixture
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11. H
Classification of chromatography :

12. CLASSIFICATION OF
CHROMATOGRAPHY
• There are two classification schemes:
Mobile phase
• Attractive forces
1. Mobile phase :
 gas (GC)
 water (LC)
 organic solvent
 supercritical fluid (SCFC)

13. CLASSIFICATION OF
CHROMATOGRAPHY
Onthebasisof
attractiveforces
adsorption
Ion exchange
Partition
Size exclusion
Gas
chromatography
Gas –solid
Gas liquid
Liquid
chromatography
Column (gravity flow)
High performance
(pressure flow ) .Thin
layer (adsorption )

14. 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

15. 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 .
 Quantitative and qualitative analysis of solute
and the components which are recovered.
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16. PLANAR CHRAMTOGRAPHY
 Separation technique – 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 tp how strongly
they interact with the stationary phase as
compared to the mobile phase
 retention factor (RF).
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17. 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.
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18. 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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19. 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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20. 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
 volatile
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21. 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

22. LIQUID CHROMATOGRAPHY
Mobilephase–liquid
 Column or a plane
 Very small packing and a relatively high pressure –high
performance liquid chromatography (HPLC)
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23. SOLVENT TYPE USED IN
LIQUID
CHROMATOGRAPHY
 ORGANIC
 Methanol
 Acetonitrile
 THF
 Methylene chloride
 Hexane
 AQUEOUS -Water
 SOLVENT MIXTURES
 Chcl3/ETOH
 MeOH/CAN /h2o
 Hexane /IPA
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24. 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 phase accounts for the separation of
different solutes .
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25. 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 .
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26. 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.
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27. THANK YOU
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