CHEM 210

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:

➢ Thin layer chromatography (TLC)

➢ Column chromatography

➢ Gas- liquid chromatography (GC)

➢ High performance liquid chromatography

(HPLC)

Modern instruments enable the use of

chromatography to determine the amount of
any component in a mixture as absolute
amount or relative to another component
(HPLC/ GC etc.) HPLC
 Thin Layer Chromatography

(TLC) is the separation of moderately volatile or nonvolatile

substances based on the differential adsorption on an inert
solid (the stationary phases) immersed in an organic solvent
or solvent mixture (the mobile phase).

➢ The stationary phase: is a polar adsorbent such as silica gel

(SiO2) or alumina (Al2O3) which has been coated into a glass,
metal or a plastic plate.

➢ The mobile phase: is an organic solvent or mixture of

solvents.
 General Procedure
➢ A small amount of the mixture to be analyzed is
spotted near the bottom of a TLC plate

➢ The TLC plate is then placed in a shallow pool of a

solvent in a developing chamber so that 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 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.
➢ The components are distributed between the
stationary phase and the solvent depending on the
polarities of the compound and the solvent.

➢ The compounds are carried up the plate (ascending

chromatography) at a rate dependent upon the nature
of the compounds and the solvent.

➢ Polar compounds are strongly attached to and held by

a polar adsorbent. Non polar compounds are held
weakly.

➢ When a non polar solvent is passed through the

adsorbent, non polar compounds are released easily,
but polar compounds are retained.
 Polarity of solvents used for adsorption
chromatography
Methanol (CH3OH) Most polar
Ethanol (CH3CH2OH)
Acetone (CH3COCH3)
Ethyl acetate (CH3CO2CH2CH3)
Methylene chloride (CH2Cl2)
Chloroform (CHCl3)
Diethyl ether (CH3CH2OCH2CH3)
Toluene (C6H5CH3)
Cyclohexane (C6H12)
Hexanes (C6H14 isomers) Least polar

➢ It is essential to find a solvent where the compound

does not run too slow or too fast on the TLC.
➢ When finding one appropriate solvent is not possible,
use a mixture of solvents of different polarity strength.
 Calculation of Rf values
The distance traveled by each component is expressed as a rate,
retention factor, or ratio of fronts Rf

distance travelled by the compound

Rf =
distance travelled by the solvent

The Rf value depends on several variables:

▪ The thickness of the adsorbent
▪ The nature of the stationary phase and its degree of
activation
▪ The mobile phase
▪ The amount of material applied
 Calculation of Rf values
Rf values are measured from the origin where the initial spot
was applied to the center of the spot.

All Rf values lie between 0 and 1.

The less polar or more soluble a compound in the mobile phase the
higher its Rf value.
Note: Chemists prefer solvent(s) that result in Rf value of 0.4 – 0.5 (0.3 -
0.7 is an acceptable range).
 Technique of Thin Layer Chromatography

1. Prepare a development chamber:

The development chamber can be a beaker covered by a watch
glass or a jar with a screw top cap.
➢ Add solvent (0.5 cm high)
➢ Line the chamber with a filter paper
➢ Put watch glass or cover on top and allow
For its saturation.

The chamber should stand

undisturbed for 5-10 minutes
to saturate the chamber
with solvent vapors.
2. Spot the TLC plate
Use a capillary tube
Mark the base line

Too much sample on a

TLC (tailing)
3. Develop the TLC plate
➢ Place the plate carefully into the
development chamber
➢ Cover the chamber with a watch glass and
leave undisturbed.
➢ Let the eluent rise until 1cm from the top.
➢ Take the plate out carefully
and immediately mark the solvent
front.

➢ Allow the solvent to evaporate completely from the plate.

4. Visualization:
➢ If the spots are colored, simply mark them with a pencil.

➢ If not, use an iodine chamber, UV light or

indicator spray.

Summary of TLC Procedure

 Co-spoting
To compare two compounds if they are the same,
➢ run a TLC of both of them side by side
➢ run by their side a “co-spot” of both compounds where the spots of the
different compounds are placed on top of one another.
➢ If the two compounds are the same, the spot of the first compound, the
second, and the co-spot would run simultaneously and the Rf values would
be the same
➢ if they are different, the two compounds would have different Rf values
and the co-spot would run into two different spots.

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.

Uneven Advance of Solvent Front:

Consequences would be inaccurate Rf values
This uneven advance can be caused by a few
factors listed below:
➢ No flat bottom.
➢ Not enough solvent
➢ Plate is not cut evenly
 Basic TLC Tips

✓ Handle TLC plates only by the edges

✓ Keep the solvent chambers capped or covered
✓ The spots should be above the solvent level in the
chamber
✓ All marks should be made in pencil
✓ Do not scratch the plates
✓ Do not leave the plates in the chamber after the solvent
has reached the top
✓ Do not move the chamber during developing
✓ Let the solvent evaporate before visualizing
 Uses of TLC
➢ To determine the purity of a compound
➢ To establish that two compounds are identical
➢ To determine the number of components in a mixture
➢ To monitor the progress of a reaction; to evaluate how
far a reaction has proceeded
➢ To determine the appropriate solvent for a column
chromatography
➢ To check the effectiveness of a separation achieved by
column chromatography, by crystallization or by
extraction
 Column Chromatography
Column chromatography is used as a purification technique: it
isolates desired compounds from a mixture.
Column chromatography works like TLC except that:

➢The stationary phase

silica gel (SiO2) or
alumina (Al2O3) is
placed in vertical glass
columns.
➢The solvent (the
eluent) which is the
mobile phase is passed
through the adsorbent
holding the mixture from
the top of the column
(descending
chromatography).
➢ The sample is loaded at the top of an adsorbent in the column.
➢ The solvent is drained through the column with the components of
the sample mixture being eluted from bottom of the column.
➢ The relative speeds that components travel are similar to their
relative Rf values in TLC.
➢ Nonpolar components travel down the column faster than polar
components and are eluted first.
➢ Normally elution will begin with a nonpolar or low polarity solvent,
then the polarity of the solvent is slightly increased.
 Two categories of column chromatography,
depending on how the solvent flows down

➢ If the solvent is allowed to flow down the column by

gravity, or percolation, it is called gravity column
chromatography.

➢ If the solvent is forced down the column by positive air

pressure, it is called flash chromatography.
 Experimental Procedure
Part 1: Thin Layer Chromatography TLC
➢ Obtain 3 TLC plate, 3 capillary tube and three colored solution
samples which are numbered
➢ Record the numbers of the vials in your report.
The colored samples are either o-nitroaniline, or p-nitroaniline and
a mixture of both which you have to identify at the end of the
experiment.
➢ Prepare the TLC for the three solutions according to the
procedure in the manual
➢ Develop the plates in 3 different solvents
1. Hexane
2. Hexane: ethyl acetate (90:10)
3. Ethyl acetate.
1. Determine which vial contains one compound and which is
a mixture of the two compounds.
2. Calculate the Rf values for the components in the 3TLC
plates
3. Identify the composition of the component/s in each vial.
4. Determine the best solvent to run the column
chromatography to separate a mixture of o-nitroaniline and
p-nitroaniline
 Part 2: Column Chromatography of an Impure Solution
➢ Obtain a clean and dry buret.
➢ Place a piece of cotton wool at the bottom.
➢ Add 0.2 cm of sand on top of the cotton in an
even way.
➢ Weigh 6 g of silica and add it on top of the Sand
sand in portions with tapping of the buret after
every addition to ensure good packing.
Silica or
➢ Add 0.2 cm of sand to the top. Alumina
➢ Connect the column to a clamped suction
flask and apply vacuum Sand
➢ Pre-elute the column by adding hexane to
Cotton
the top of the column (around 6 mL) opening
wool
the stopcock to let the solvent move to the
bottom. to aspirator
➢ Follow the procedure in the
manual to separate the mixture of ortho and
para nitroaniline
Figure 4.6
Notes: 1. Avoid air bubbles in the column
2. Never allow the column to run dry! (cracking)
Perform TLC procedure on the collected fractions:

➢ Using new TLC plates, perform TLC for the collected

fractions and the pure solutions that you have, using the
solvent chosen to run the column.

➢ Determine if the collected fractions are pure.

➢ Determine the identity of the collected samples by

comparing the location of the spots in the collected samples
to those of the pure samples