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210 - Chromatography - Sp. 2023
210 - Chromatography - Sp. 2023
Chromatography
Experiment 4: Chromatography
Lecture Outline
1. Purpose of the Experiment
2. Theory of Chromatography
3. Types of Chromatography
4. Thin Layer Chromatography TLC
5. Solvents Used in Adsorption
Chromatography
6. Calculation of Rf Values
7. Technique of TLC
8. Uses of TLC
9. Column Chromatography
10. Procedure
Purpose
1. To learn the principles of chromatography
2. To learn the technique of thin layer chromatography
(TLC)
3. To calculate the Rf value of o-nitroaniline and p-
nitroaniline, in three different solutions.
4. To determine the best solvent to run a column
chromatography in order to separate a mixture of o-
nitroaniline and p-nitroaniline
5. To identify a compound in a mixture by comparing its Rf
value with the Rf of the known compound.
Theory
Chromatography is a method for the separation of a mixture
of compounds based on the different partitioning or
distribution of the compounds between a stationary phase
and a mobile phase.
➢ Chromatographic techniques are used extensively in the
organic lab for both qualitative separations and quantitative
analysis.
➢ All chromatographic techniques are based on a similar
principle:
Components of a mixture can often be differentiated by
exposure to two competing phases;
- the stationary phase and
- the mobile phase.
Types of Chromatography
Adsorption Chromatography: The stationary phase is a solid and
the mobile phase is a liquid or a gas. Mixture components are
adsorbed on the surface of the stationary phase with different powers
that account for separation.
Partition Chromatography: The stationary phase is a liquid forming
a thin film on an inert solid acts as support. The mobile phase is a
liquid or a gas.
Ion Exchange Chromatography: It is used for separation of charged
molecules. The stationary phase is an ion exchange resin to which a
cationic or anionic groups are covalently bonded. Solute ions of the
opposite charge are attracted to the stationary phase. The mobile
phase is a liquid.
Size Exclusion Chromatography: method in which molecules in
solution are separated by their size.
Electrophoresis: is a method used to separate proteins by charge
and or size.
Mechanisms of Separation
Most common methods of chromatography:
➢ Column chromatography
c c
b b
a a
A A/B B A A B
A/B B AA BB A A B B A B
If A and B are different, the co-spot of A and B will appear as two spots
having the same Rf values of A and B.
Common Problems in TLC
Over-large Spots: Spotting sizes of your
sample should not be larger than 1-2 mm in
diameter. An over-large spot could cause
overlapping of other component spots with
similar Rf values on your TLC plate, it would
prove difficult to resolve the different
components.