Chromatography

Chromatography is a technique used to separate substances into their

components. It has a stationary phase ( a solid or liquid supported on a solid) and a
mobile phase (liquid or gas). The mobile phase flows through the stationary phase
and carries the components of the mixture with it. Different components travel at
different rates.
In paper chromatography, the stationary phase is a very uniform absorbent
paper. The mobile phase is a suitable liquid solvent or mixture of solvents.
A distinct advantage of TLC is the very small quantity of sample required. A
lower limit of detection of 10-9 g is possible in some cases.
(http://chemistry.csudh.edu/faculty/noel/CHE317L/Thin%20Layer
%20Chromatorgaphy%20Experiment.htm)
Technique
In this experiment, 2 known solutions were used to contrast a third, unknown
solution. First, an origin was marked with a pencil, approximately 50 mm from the
bottom of the plate. Then each solution was spotted on the plate using capillary
tubes. The third, unknown solution was spotted as well. The plate was then placed in
a beaker containing the solvent, ethyl acetate petroleum ether 60-80, making sure
the pencil mark is above the level of the developing solvent. It was then allowed to
develop in the fume cupboard until the solvent reached approximately one
centimetre from the top. This was marked again with a pencil. The plate was taken
out of the developing chamber and allowed to dry. UV light was used to mark the
development.
Separation of substances is based on the many equilibrations the solutes
experience between the moving (ethyl ethanoate) and stationary (silica) phase. Less
polar substances move more quickly than more polar ones. In general the stationary
phase is strongly polar and strongly binds polar substances. The moving liquid
phase is usually less polar than the adsorbent and most easily dissolves substances
that are less, or even non-polar. The results are made visible by UV absorption or by
chemical reaction with iodine.
(http://www.liskeard.cornwall.sch.uk/images/Liskeard-Sixth-Form/AspirinSelection.pdf)
The retardation factor was then calculated for the two known solution,
resulting in the unknown solution being paracetamol.

(SiO2.xH2O)n silica gel

There are three components in the TLC which include the TLC plate with
adsorbent, the development solvent and the organic compounds to be analysed. The
adsorbent, silica gel consists of a three dimensional network of thousands of
alternating silicon and oxygen bonds. It is a very polar and is capable of hydrogen
bonding due to its partial positive charge in silicon and partial negative in oxygen.
The silica gel will compete with the development solvent for the organic compounds
as the solvent is traveling up through the TLC plate. The silica gel tends to bind the
compounds (on stationary phase) while the development solvent tried to dissolve the
compounds (on mobile phase) in order to carry the compounds along the plate as
the solvent travels up. All the compounds are possible to be adsorbed into the
stationary phase however the time of adsorption of compounds in the particular
phase is depends on the polarity of each compound. The more polar the compound
is, the longer the time taken that the compound adsorbed into the stationary phase
so it eluting speed is slower (more time on stationary phase). Less polar compounds
are weakly adsorbed, so the time taken for less polar compounds to be adsorbed on
stationary phase is shorter. As a result, the less polar compounds can travel further
along the plate compared to the more polar compounds.
In this experiment, the analgesics drugs have been analysed by using TLC
are acetaminophen and aspirin. The structure of each compounds are shown below:

The polarity of the compounds could be compared by looking at the

structure of these compounds. The polarity of the compound is due to the
effect of electronegativity in atoms and the asymmetrical structure of
compound. The unequal sharing of electron within the bond causes the
formation of an electric dipole which leads to partial positive and partial
negative exist in the several atoms. The highly electronegative atoms present
in these compounds are O, N, and F. These highly electronegative atoms tend
to withdraw the electrons towards themselves from the aromatic ring and
hence it polarizes the compounds. Nitrogen and oxygen atom present in these
compounds have higher electronegativity compared to the carbon atom and
hydrogen atom. Thus, nitrogen and oxygen atom acquire partial negative
charge while the carbon and hydrogen atom acquire partial positive charge.
Hence, paracetamol has the highest polarity, followed by aspirin.
Based on the retardation factor, Rf, the polarities of these compounds
can be compared. The higher the Rf, the lower the polarity and hence the
distance traveled by the compound would be longer. Aspirin has the highest
Rf value. The interaction between aspirin and stationary phase (silica gel) is
the weakest which allows the aspirin can travels up along the plate fastest. A
stronger interaction in between the silica gel and acetaminophen causes the
compound move slower when travels up the TLC plate. In this part, TLC
method can be used to identify the two unknowns spotted on the TLC plate.
The compounds with same polarities usually travel up through the plate in the
same distance and possess the same Rf value provided the stationary phase
and mobile phase are identical.
Rf paracetamol = 7mm/50mm = 0.14
Rf aspirin = 30mm/50mm = 0.60
Rf unknown = Rf paracetamol