1. ADSORPTION CHROMATOGRAPHY
Muhammad Tanveer Khan

2. 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.”

3. PRINCIPLE
Principle involves competition of components of sample mixture for active site on adsorbent. These active sites are formed in molecule due to
* Cracks
* Edges
The electrostatic forces present in the molecule, which hold together the crystal lattice, are directed outward. These forces and electrostatic forces of solute molecule cause separation.

4. 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.

5. TYPES

6. 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.”

7. 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

8. 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
Instrumentation

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

10. 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.”

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

12. 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

13. 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.

14. 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

15. 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.

16. 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.

17. 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

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

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

20. PARTITION CHROMATOGRAPHY

21. 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.”

22. 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.
This gives basis for separation.

23. 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.

24. TYPES

25. PAPER CHROMATOGRAPHY
“.”

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

27. 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

28. 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

29. 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.

30. 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.

31. 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

32. Documentation
Storage of chromatogram.
Calculating Rf values

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

34. ION EXCHANGE CHROMATOGRAPHY

35. DEFINITION
In this type of chromatography, a resin (the stationary solid phase) 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.
Ion exchange mechanism separates analytes based on their respective charges.
Ion exchange chromatography is performed in columns but can also be useful in planar mode.

36. MECHANISM
Ion exchange chromatography uses a charged stationary phase to separate charged compounds including anions, cations, amino acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained.
Ion exchange chromatography is commonly used to purify proteins.

37. MECHANISM

38. SIZE EXCLUSION CHROMATOGRAPHY

39. DEFINITION
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 allows smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones.

40. It is generally a low-resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification.
It is also useful for determining the tertiary structure and quaternary structure of purified proteins.

41. MECHANISM

43. AFFINITY CHROMATOGRAPHY

44. DEFINITION This is the most selective type of chromatography.
It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase.
For example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH.