1. TOPIC: CHROMATOGRAPHY
PRESENTED BY : Aadil Ali Wani

2. Chromatography
History
Mikhail Tswett, Russian Botanist, 1872-
1919,
In 1906 Tswett used to chromatography
to separate plant
pigments. He called the new technique
chromatography because from Greek
chroma which means "color" graphein "to
write "adsorbent column

3. • Importance
Chromatography has application in
every branch of the physical and
biological sciences.
12 Nobel prizes were awarded between 1937 And 1972 alone
for work in which chromatography played a vital role.

4. • Chromatography is a physical method of
separation in which the components to be
separated are distributed between two
phases
• one of which is stationary (stationary phase)
while the other (the mobile phase) moves
through it in a definite direction.
• The chromatographic process occurs due to
differences in the distribution constant of
the individual sample components.

5. • Chromatography Is a technique used to
separate and identify the components of a
mixture.
• Works by allowing the molecules present in
the mixture to distribute themselves
between a stationary and a mobile medium.
• Molecules that spend most of their time in
the mobile phase are carried along faster.

6. Classification of chromatography according to
mobile phase:
1- Liquid chromatography: mobile phase is a
liquid. (LLC, LSC).
2- Gas chromatography : mobile phase is a gas.
(GSC, GLC).

7. • Classification according to the packing of the
stationary phase:
• 1- Thin layer chromatography (TLC): the
stationary phase is a thin layer supported on
glass, plastic or aluminum plates.
• 2- Paper chromatography (PC): the stationary
phase is a thin film of liquid supported on an
inert support.
• 3- Column chromatography (CC): stationary
phase is packed in a glass/steel column.

8. ADSORPTION CHROMATOGRAPHY
• DEFINITION
• “It is a type of chromatography in which a mobile
liquid or gaseous phase is adsorbed onto the
surface of a stationary solid phase. The
equilibration between the mobile and stationary
phase accounts for the separation of different
solutes.”

9. PRINCIPLE
• Separation occurs because of the fact that an
equilibrium is established between molecules
adsorbed on stationary phase and those
which are flowing freely in mobile phase.
• The more the affinity of the molecule of
particular component, less will be its
movement.

10. • TYPES of Adsorption chromatography
• Column chromatography
• Thin layer chromatography
• Gas solid chromatography

11. ADSORBENTS
• “An adsorbent is a substance, usually porous in
nature and with a high surface area that can adsorb
substances onto its surface by intermolecular
forces.”
• AN IDEAL ADSORBENT :- The Ideal adsorbent must
fulfill the following requirements:
• Insoluble in mobile phase
• Inert to solutes (adsorptive)
• Colorless especially when we work with colored
mixtures
• Suitable particle size enough to give good
separation and reasonable flow rate

12. COMMON ADSORBENTS
• Hydrated silica gel
• Silica gel G
• Silica gel S
• Silica gel GF254
• Silica gel H
• Silica gel N
• Silica gel HF254
• Silica gel PF254
• Modified silica gel
• Alumina
• Kieselghur (Diatomaceous earth)
• Cellulose MN300
• Cellulose microcrystalline

13. Thin layer chromatography{(tlc}
• is a method for identifying substances and
testing the purity of compounds.
• TLC is a useful technique because it is relatively
quick and requires small quantities of material.
• “The technique which involves flowing of mobile
phase over a thin layer of adsorbent, applied on
solid support, where separation of components
occur by differential migration which occurs
when solvent flows along fine powder spread on
glass plates, is called thin –layer
chromatography.”

14. Instrumentation
• Chromatography jar
• Capillary tube
• Thin layer chromatography plate
• Stationary phase
• Mobile phase

15. • Chromatography jar: It is made of glass
and has a lid on it. Jar maintains proper
environment that is required for
separation.
• Capillary tube: It is used to apply sample
mixture on TLC plate.
• Stationary phase: Adsorbents

16. • Separations in TLC involve distributing a mixture
of two or more substances between a stationary
phase and a mobile phase.
• The stationary phase:
• is a thin layer of adsorbent (usually silica gel or
alumina) coated on a plate.
• The mobile phase:
• is a developing liquid which travels up the
stationary phase, carrying the samples with it.
• Components of the samples will separate on the
stationary phase according to how much they
adsorb on the stationary phase versus how much
they dissolve in the mobile phase.

17. • Instrumentation Mobile phase:
• Mobile phase may be a single liquid or a
mixture of liquids.
• Commonly used mobile phases are;
• •Methanol
• •Ethanol
• •Ethyl acetate
• •Diethyl ether
• •Acetone
• •Chloroform
• •Hexane

18. Procedure
• Clean and dried chromatography jar is taken.
• A paper impregnated in the mobile phase is applied to the
walls to ensure that atmosphere of the jar is saturated with
solvent vapors.
• Mobile phase is added to the jar at a length of 0.5-1cm from
the bottom.
• Jar is closed.
• Equilibrium is allowed to be maintained.
• Base line is marked on adsorbent.
• Sample is applied on TLC plate with help of capillary tube.
• Sample spot is air dried.
• TLC plate is put in the chromatography jar and lid is closed.
• The system is allowed to be static until the solvent moves to a
proper distance from baseline.
• TLC plate is taken out and dried

19. Preparing the Plates for Development
• With a pencil, etch two small notches into the adsorbent about
2 cm from the bottom of the plate.
• The notches should be on the edges of the plate, and each
notch should be the same distance up from the bottom of the
plate.
• The notches must be farther from the bottom of the plate than
the depth of the solvent in the jar.
• Using a drawn-out capillary tube, spot the samples on the
plate so that they line up with the notches you etched.

20. Location of separated components
• If the sample is separated into colored
components, then the location is dried in
ordinary light. But in case of colorless
components following are used
• •UV lamp
• •Iodine crystals
• •Spraying agents

21. Thin Layer Chromatography (TLC)

22. Applications
• It is used for separation and identification of;
• Amino acids
• Peptides and proteins
• Alkaloids
• Carbohydrates
• Fats and fatty acids
• Antibiotics
• Narcotic analgesics
• Glycosides

23. Summary
• A TLC plate is a sheet of glass, metal, or plastic which is coated
with a thin layer of a solid adsorbent (usually silica or
alumina).
• A small amount of the mixture to be analyzed is spotted near
the bottom of this plate.
• The TLC plate is then placed in a shallow pool of a solvent in a
developing chamber so that only the very bottom of the plate
is in the liquid.
• This liquid, or the eluent, is the mobile phase, and it slowly
rises up the TLC plate by capillary action.
• As the solvent moves past the spot that was applied, an
equilibrium is established for each component of the mixture
between the molecules of that component which are
adsorbed on the solid and the molecules which are in
solution.

24. • In principle, the components will differ in
solubility and in the strength of their adsorption
to the adsorbent and some components will be
carried farther up the plate than others.
• When the solvent has reached the top of the
plate, the plate is removed from the developing
chamber, dried, and the separated components
of the mixture are visualized.
• If the compounds are colored, visualization is
straightforward. Usually the compounds are not
colored, so a UV lamp is used to visualize the
plates.

25. Paper Chromatography
• A method of partition chromatography using filter
paper strips as carrier or inert support.
• The factor governing separation of mixtures of
solutes on filter paper is the partition between two
immiscible phases.
• One is usually water adsorbed on cellulose fibers in
the paper (stationary phase).
• The second is the organic solvent flows past the
sample on the paper (stationary phase).
• Partition occurs between the mobile phase and the
stationary aqueous phase bound by the cellulose.
• The isolation depends on partition coefficient of the
solute.

26. Instrumentation
Chromatography jar
Capillary tube
Stationary phase (liquid impregnated paper)
Mobile phase
Chromatography jar:
It is made of glass and has a lid on it. Jar maintains proper
environment that is required for separation.
Capillary tube:
It is used to apply sample mixture.
Stationary phase:
liquid impregnated paper

27. • Mobile phase:
• Mobile phase may be a single liquid or a mixture of
• liquids.
• Commonly used mobile phases are;
• •Methanol
• •Ethanol
• •Ethyl acetate
• •Diethyl ether
• •Acetone
• •Chloroform

28. Procedure
• Clean and dried chromatography jar is taken.
• A paper impregnated in the mobile phase is
applied to the walls to ensure that atmosphere
of the jar is saturated with solvent vapors.
• Mobile phase is added to the jar at a length of
0.5-1cm from the bottom.
• Jar is closed.
• Equilibrium is allowed to be maintained.
• Base line is marked on adsorbent.

29. • Sample is applied on paper with help of capillary
tube.
• Sample spot is air dried.
•
• Paper is put in the chromatography jar and lid is
closed.
• The system is allowed to be static until the
solvent move to a proper distance from baseline.
• Paper is taken out and dried.

30. Location of separated components
• If the sample is separated into colored
components, then
• the location is dried in ordinary light. But in
case of
• colorless components following are used;
• •Uv lamp
• •Iodine crystals
• •Spraying agents

31. Documentation
• Storage of chromatogram.
• Calculating Rf values

32. Applications
• It is used for separation and identification of;
• Amino acids
• Carbohydrates
• Tannins
• Glycosides
• Alkaloids etc.

33. • Modern applications of chromatography
• creating Immunization
• Food testing
• Brewage testing
• drug testing
• Forensic testing
• Green chemistry
• Chiral separation (teratogens :thalidomide :birth
defects

34. .
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