Chromatography; history and its types.

1. By
Group 2

2. Contents
• Introduction
• History
• Principle
• Types of chromatography
• Conclusion
• References

3. Introduction
By
Zeeshan Khalid
Reg# 634/BSBT/F14

4. Introduction
• Chromatography
• Chroma Color
• Graphein To write

5. Chromatography
• Chromatography is used to separate mixtures
of substances into their components.

6. History
• It was first employed in Russia by
the Italian- scientist Mikhail
Tsvet in 1900.
• The separation of plant pigments
such as Chlorophyll.
• New types of chromatography
developed.

7. Principle
• All forms of
chromatography work on
the same principle.
• Stationary phase (a solid,
or a liquid supported on a
solid)
• Mobile phase (a liquid or a
gas)

8. High performance liquid
chromatography
Asfand Yar Khan
Reg# 635/BSBT/F14

9. Introduction
• HPLC is a form of liquid chromatography used to separate
compounds that are dissolved in solution.
• HPLC instruments consist of a reservoir of mobile phase, a pump,
an injector, a separation column, and a detector.
• Compounds are separated by injecting a sample mixture onto the
column.
• The different component in the mixture pass through the column at
differentiates due to differences in their partition behavior between
the mobile phase and the stationary phase.
• The mobile phase must be degassed to eliminate the formation of air
bubbles.

10. What is Liquid Chromatography?
• Liquid chromatography is a separation technique that involves:
• the placement (injection) of a small volume of liquid sample
• into a tube packed with porous particles (stationary phase)
• where individual components of the sample are transported along the
packed tube (column) by a liquid moved by gravity.
• The components of the sample are separated from one another by the
column packing that involves various chemical and/or physical interactions
between their molecules and the packing particles.
• The separated components are collected at the exit of this column and
identified by an external measurement technique , such as a
spectrophotometer that measures the intensity of the color , or by another
device that can measure their amount.

11. Principles of Liquid Chromatography

12. Principles of HPLC
Principle;
Tabel shows the relationship b/w various parameters
of HPLC
Trendline:
• Stationary phase have small particulate size and high
surface areas.
• Columns: 20 cm or less
• Mobile phase pumped at high pressures of 200Bar,
3000 psi.
• Flow rates: 1-3 cm3 per min
Column length No. of theoretical
plates per unit area
Resolving power Column length
Particle size Surface area

13. What is HPLC?
• HPLC is a separation technique that involves:
•the injection of a small volume of liquid sample
•into a tube packed with tiny particles (3 to 5 micron ( μm ) in diameter
called the stationary phase)
•where individual components of the sample are moved down the
packed tube (column) with a liquid (mobile phase) forced through
the column by high pressure delivered by a pump.
• These components are separated from one another by the column packing
that involves various chemical and/or physical interactions between their
molecules and the packing particles.
• These separated components are detected at the exit of this tube (column) by a
flow-through device (detector) that measures their amount. An output from
this detector is called a “liquid chromatogram”.

14. Gas chromatography
Muhammad Jawad
Reg # 636/BSBT/F14

20. Ion exchange chromatography
Zeeshan Khan
Reg # 637/BSBT/F14

21. inTro
 This technique was developed in 19th century and was used to
purify the drinking water.
 Reversible exchange of ions in the solution with ions
electrostatically bound to some sort of insoluble matrix or
stationary phase
.
 It is extremely useful in the separation of charged compounds
like proteins differing from only one charged amino acid.
 Ion exchanged is performed in the column.

22. Principle
The principle of separation is by reversible
exchange of ions between ions present in the
solution and those present in the ion exchange
resin.

23. Ion exchangers
There are two types of ion exchangers.
Cations exchanger.
Anions exchanger.

24. Cation exchange chromatography
Positively charged molecules are attracted to a
negatively charged solid support. Commonly used
Cation exchange resins are S-resin, sulfate
derivatives; and CM resins, carboxylate derived ions

25. Anion exchange chromatography
Negatively charged molecules is attracted to a
positively charged solid support. Commonly used
anion exchange resins are Q-resin, a Quaternary
amine; and DEAE resin, Di-EthylAminoEthane.

26. CATION AND ANION
EXCHANGER

27. Uses
Used in the analyses of aminoacid
To determine the base composition of the nuclic
acid.
Proteins are also successfully seprated by this
technique
Used for sepration of vitamens
This is most effective method for water
purification. Complete deionization of water (or)
a non-electrolyte solution is performed by
exchanging solute cations for hydrogen ions and
solute anions for hydroxyl ions. This is usually
achieved by method is used for softening of
drinking water.

28. Thin layer chromatography
Hammad Ali
Reg # 638/BSBT/F14

33. Paper chromatography
Muzaffar Latif
Reg # 639/BSBT/F14

34. Definition
• Paper chromatography is a type of chromatography.
• It is an analytical method used to separate colored chemicals
or substances.
• A method of separating components of a mixture by
differential movement through a two-phase system: the
mobile phase and the secondary phase.
• The separation of an unknown substance is mainly carried
out by the flow of solvents on the specially designed
chromatographic paper

35. Principle
• Paper chromatography works on the principle of partition, i.e., it
is based upon continuous differential distribution of the various
components of the mixture between the stationary and the mobile
phase.
• Cellulose layers in filter paper contains moisture which acts as
stationary phase & organic solvents/buffers are used as mobile
phase.
• In paper chromatography the results are represented by Rf value
which represents the movement of component of the mixtures
relative to the solvent front.
Rf value = Distance traveled by the solute
Distance traveled by the solvent

36. Procedure
Solvent
front
Filter
paper
Ink spots
Solve
nt
Beaker
Separated
dyes

37. Observing the Chromatograms
Concentration of
Isopropanol
0
%
20
%
50
%
70
%
100
%

38. Uses of Paper chromatography
• To Analyze – examine a mixture, its components, and their
relations to one another
• To Identify – determine the identity of a mixture or
components based on known components
• To Purify – separate components in order to isolate one of
interest for further study
• To Quantify – determine the amount of the a mixture and/or
the components present in the sample

39. Column chromatography
Muhammad Sharif
Reg # 640/BSBT/F14

40. Column chromatography
An impure sample is loaded onto a column of
adsorbant, such as silica gel
. An organic solvent or a mixture of solvents (the
eluent) flows down through the column.
Components of the sample separate from each
other by partitioning between the stationary
packing material.

41. Packing a (silica gel) column:
• 1- Add cotton to bottom of column.
• 2-Add sands to the column
• 3- Prepare a slurry of silica in the intial eluent by
pouring dry silica into a beaker of eluent.
• 4-Quickly but carefully pour the slurry into the
column.
• 5-Drain eluent from the column until no solvent
remains .
• 6- Loading a sample onto the column

42. Column

43. Precautions

44. Affinity chromatography
Umer Awais
Reg # 641/BSBT/f14

45. Affinity Chromatography
Affinity chromatography is one of the most diverse and
powerful chromatographic methods for purification of a
specific molecule or a group of molecules from complex
mixtures.

46. Principle
Affinity chromatography is based on highly specific biological
interactions between two molecules, such as interactions
between enzyme and substrate, receptor and ligand, or
antibody and antigen.
These biological interactons are typicaly reversible these
reversible interactions are used for purification .

47. Procedure
Purification is done by placing one of the interacting molecules,
referred to as affinity ligand, onto a solid matrix to create a
stationary phase while the target molecule is in the mobile
phase. Successful affinity purification requires a certain
degree of knowledge and understanding of the nature of
interactions between the target molecule and the ligand to help
determine the selection of an appropriate affinity ligand and
purification procedure.

50. Conclusion
• Separation technique
• Many types of chromatography
• Working depend on stationary and mobile
phase
• Many applications in the field of biochemistry,
organic chemistry, etc

51. Thanks