Chromatography by Dr. Tahir ALi Pharmacognosy department, RIPS, Riphah International University.

1. Chromatography
Dr. Tahir Ali
RIPS
Riphah International University

2. For more detail follow the link: https://www.youtube.com/watch?v=e8i374hrWQg

3. What is Chromatography?
Chromatography, literally "color writing", was first employed by
Russian scientist Mikhail Tsvet in 1900.

4. DEFINITION
• “ It is a physical separation method in
which the components of a mixture are
separated by differences in their
distribution between two phases, one of
which is stationary (stationary phase)
while the other (mobile phase) moves
through it in a definite direction . The
substances must interact with the
stationary phase to be retained and
separated by it .
What is Chromatography?

5. What is Chromatography?
Chromatography is a technique for separating mixtures
into their components in order to analyze, identify,
purify, and/or quantify the mixture or components.
Separate
• Analyze
• Identify
• Purify
• Quantify
ComponentsMixture

6. Applications of Chromatography
Forensics
Research Pharmaceutical industry

7. Types of Chromatography…
Paper
HPLC Gas
Thin layer
Column

8. CLASSIFICATION
According to mechanism of separation
1. Ion-exchange chromatography
2. Affinity chromatography
3. Size-exclusion chromatography
4. Adsorption chromatography
5. Partition chromatography

9. ION-EXCHANGE CHROMATOGRAPHY
DEFINITION:
• Ion-exchange chromatography (or ion
chromatography) is a chromatography process that
separates ions and polar molecules based on their
affinity to the ion exchanger. It works on almost any
kind of charged molecule—including large proteins,
small nucleotides, and amino acids.
• For more detail visit the following links:
– https://www.youtube.com/watch?v=efUrl_djzQ0
– https://www.youtube.com/watch?v=q3fMqgT1do8

11. • If the stationary phase is represented by R− or R+
and the sample by X+ and X−, retention in IEC can
be represented as
X+ + R−K+ X+R− + K+ (cation exchange)
X- + R+Cl- X-R+ + Cl- (anion exchange)

12. APPLICATIONS:
It can be used for almost any kind of
charged molecule including large
proteins, small nucleotides and amino
acids.
1. Protein purification
2. Water analysis
3. Quality control

13. AFFINITY CHROMATOGRAPHY
• It is a method of separating
biochemical mixtures based on a
highly specific interaction such as
that between antigen and antibody,
enzyme and substrate, or receptor and
ligand.

14. APPLICATIONS:
• Purify and concentrate an enzyme
solution
• Purification of recombinant proteins
• Purification of antibodies

15. SIZE-EXCLUSION CHROMATOGRAPHY
• It is also known as gel permeation or gel filtration
chromatography.
• This type of chromatography lacks an attractive
interaction between the stationary phase and
solute. The liquid or gaseous phase passes
through a porous gel which separates the
molecules according to its size. The pores are
normally small and exclude the larger solute
molecules, but smaller molecules are able to
enter the pores of the media and, therefore,
molecules are trapped and removed from the flow
of the mobile phase. This causes the larger
molecules to pass through the column at a faster
rate than the smaller ones

16. SIZE-EXCLUSION CHROMATOGRAPHY

17. APPLICATIONS:
• Purification and analysis of synthetic and
biological polymers, such as;
– Proteins, Polysaccharides, Nucleic acids.
• It is also useful for determining the
tertiary structure and quaternary
structure of purified proteins.

18. ADSORPTION
CHROMATOGRAPHY

19. 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.”

20. 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.

21. TYPES
Adsorption
chromatography
Column
chromatography
Thin layer
chromatography
Gas solid
chromatography

22. 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.”

23. AN IDEAL ADSORBENT
The Ideal adsorbent must fulfill the following
requirements:
Insoluble in mobile phase
Inert to solutes (adsorptive)
Colorless especially when work with
colored mixtures
Suitable particle size enough to give good
separation and reasonable flow rate

24. 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

25. COMMON ADSORBENTS
Modified silica gel
Alumina
Kieselghur (Diatomaceous earth)
Cellulose MN300
Cellulose microcrystalline

26. THIN-LAYER CHROMATOGRAPHY
“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.”

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

28. Instrumentation
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

29. Instrumentation
TLC plate:
Borosilicate glass plates are preferred. Most commonly
used
sizes are;
• 20 X 20cm
• 20 X 10cm
• 20 X 5cm
Microscopic slides are also used.

30. 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

31. 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.

32. Procedure
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 move to a proper distance from
baseline.
TLC plate is taken out and dried.

33. 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

34. Documentation
• Storage of chromatogram for TLC is difficult. It is usually
undesirable since plates are employed for repeated use.
Various methods for documentation include;
Rf value in TLC
Preservation of chromatogram by peeling off
adsorbent.
Graphical copying i.e. tracing on transparent
paper.
Photography

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

36. PARTITION
CHROMATOGRAPHY

37. DEFINITION
“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 between the mobile
phase and the stationary liquid.”

38. PRINCIPLE
• Separation of components of a sample mixture
occurs because of partition.
• Stationary phase is coated with a liquid which is
immiscible in mobile phase.
• Partition of component of sample between sample
and liquid/ gas stationary phase retard some
components of sample more as compared to others.

39. PRINCIPLE
The stationary phase immobilizes the liquid
surface layer, which becomes stationary phase.
Mobile phase passes over the coated adsorbent
and depending upon relative solubility in the
coated liquid, separation occurs. The component
of sample mixture appear separated because of
differences in their partition coefficient.

40. TYPES
Partition
chromatography
Liquid-liquid
chromatography
Gas-liquid
chromatography

41. PAPER
CHROMATOGRAPHY

42. Instrumentation
Chromatography jar
Capillary tube
Stationary phase (liquid impregnated paper)
Mobile phase

43. Instrumentation
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

44. 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

45. 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.

46. Procedure
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.

47. 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

48. Documentation
Storage of chromatogram.
Calculating Rf values

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