P6 P5 M3 D3

3
Chromatography Practical Assignment

Chromatography is a technique that helps identify the components of a mixture by separating

the substances. The mixture is spread out across a surface known as the "stationary phase,"
which could be silica gel, paper or other porous solids. After that, it is put into a liquid or gas
that is referred to as the "mobile phase"; this is done to ensure that the substance is taken with
the mobile phase as it progresses up the stationary phase. The type of material used for the
stationary phase determines how the solute behaves in the mobile phase; possible forms of
adhesion include partition, often referred to as absorption, and adsorption.

Adsorption chromatography uses a solid stationary phase and a liquid or gaseous mobile
phase. Adsorption is the process by which one material adheres to the surface of another (the
mixture's components to the stationary phase). The equilibrium between adsorption onto the
solid surface and solubility in the solvent varies for each component (solute); the less soluble
or "best absorbed" components adhere more to the stationary phase and move more slowly.
This leads to a separation into bands with various solutes in them.
Alumina and silica gel are two examples of stationary phases used in adsorption
chromatography.

Partition, additionally referred to as absorption, is a kind of chromatography in which an inert

solid's surface is coated with a thin layer (or film) of a non-volatile liquid as the stationary
phase – this is known as Thin Layer Chromatography (TLC for short). With more soluble
components in the mobile phase travelling quicker, the mixture to be separated is transported
by a gas or liquid as the mobile phase and the solutes disperse themselves between the
moving and the stationary phases. Partition chromatography includes paper chromatography.

During practical work, my class completed 3 chromatographs, using both adsorption and
partition chromatography.

Paper chromatography (amino acids)

The first chromatograph used partition chromatography - in order to determine which amino
acids were in a mixture, we applied the mixture of amino acids alongside the various
separate amino acids. The stationary phase in this partition chromatography technique was
paper. Since ammonia is harmful if inhaled, the experiment had to be carried out in a fume
cupboard because the mobile phase consisted of a mixture of ammonia, ethanol, and water.
The substances in the mobile phase mix are all polar, particularly water which is very polar –
this is due to the unsymmetrical shape of the molecule and the unequal sharing of electrons
between the atoms in the compound. In This mixture was picked for the chromatograph
because all of the amino acids we used are also polar, and the polar molecules will be more
attracted to a polar solvent, meaning they will travel up the stationary phase and stick.
order to prevent the amino acids from touching the solvent, we first drew a line 2 cm from the
bottom of the paper (in pencil because it is not soluble), then evenly spaced 5 markings along
the paper to indicate the locations where the amino acids (and mix) would be dropped. We
used a dropper to place each amino acid on its designated mark after labelling the following:
glutamic acid, leucine, aspartic acid, lysine, and the mixture in the middle. We then rolled the
paper so that one edge touched the other and fastened it with paper clips so that it would sit
upright in the beaker of the solvent. We put the paper inside the fume hood, covered it with
cling film to prevent the ethanol and ammonia from escaping, and then we waited.
After the solvent (mobile phase) had completed its ascent up the paper, we took it out of the
beaker and marked the solvent's location with a line (referred to as the solvent front, which is
used to determine Rf values). After that, the paper was let to dry.
The prep room workers then applied a solution known as ninhydrin, which interacts with the
amino acids to make them appear purple so we can see them -this is called a revealing agent.
Due to the solvent's high polarity and the attraction between polar and polar molecules, the
components travel different distances and separate for different reasons. The higher the
polarity, the more attracted the molecules in the components are to the molecules in
the solvent causing them to stay together. And the more the component follows the solvent as
it moves up the stationary phase, the higher the component travels up the paper.
Once we had our results in front of us, we calculated the distance travelled by
each component as well as the distance from the start line to the solvent front.

Amino Acid
Partition
Chromatograph

We used the
formula Rf =
Distance Travelled
by Solute/Solvent
Front to get the Rf values of each amino acid. In order to determine which amino acids were
present in the mixture, we also calculated the Rf values of each component that had been
isolated from it and compared them to the RF values of the individual amino acids.

The results I got for the Rf values were: 0.4 (Glutamic Acid), 0.95 (Leucine), 0.44 (Aspartic
Acid) and 0.47 (Lycine). From these results, we deduced that the mixture contained Leucine,
Aspartic Acid and Lycine.
Overall, I don't think my experiment worked well; a lot of the amino acids didn't migrate up
the stationary phase and stop at the appropriate locations, and my mixture didn't separate
completely, which made it difficult to interpret. If I were to repeat the experiment, I would
make sure to use just one drop of the amino acids and to apply it lightly in order to avoid
adding more than is necessary—just because I can't see it doesn't mean there is not enough.
Additionally, I might work in a sterile setting and use gloves to prevent contamination
resulting in smears and fingerprints that would make it more difficult to read the
chromatograph.

Plant pigments
We used similar techniques for the other two chromatographs; however, we were
investigating the different components in plant pigments.

Thin Layer Chromatography (Plant pigments)

One of the chromatographs used TLC which is a type of adsorption chromatography and used
silica gel as the stationary phase and toluene as the solvent (mobile phase). Unlike the solvent
used for the amino acids, toluene is a non-polar solvent, it was used because the plant
pigments are also non-polar, so they are attracted to the non-polar solvent. As with the Amino
Acid chromatograph, we drew a line 2cm up from the bottom of the stationary phase and then
smeared the plant pigments along the line. The solvent that was used for this chromatograph
is a substance called toluene. The next step was to place it into the beaker of toluene, this was
also done in the fume hood to protect us from fumes. We then waited for the pigments to be
taken up the stationary phase until they were all separated.

Peak fronting, an error in glutamic acid, and peak tailing in lysine are caused by the solute
moving some molecules more quickly than others and by adding too much solute. As you can
see in the chromatograph, the Lycine and Aspartic Acid also split into two separate parts,
which meant that some molecules went higher up the stationary phase than others. I believe
that this was caused by contamination and adding too much solute. The molecules that go
higher up the stationary phase are the anomalous results and are considered "mistakes”.

We measured the Rf values for each of the six distinct substances that the plant pigments had
separated into after drawing a line along the solvent front and letting them dry. Then, in order
to determine whether they were conclusive, this was compared to our controls. My results
were not entirely definitive although they were quite close, even if they weren't absolute. This
chromatograph might not be entirely conclusive for a number of reasons, such as
contamination or the addition of either too little or too much solute. Though I don't think any
of these specific mistakes happened with this chromatograph, I do believe that keeping the
solute on the stationary phase for an extended period of time before adding solvent could
have been a problem. We had to wait until it was our turn to put the column—which was a
single line of plant pigments along the silica gel—into the beaker of toluene since there
wasn't enough to put the chromatographs into. This indicates that many of the molecules were
already attached to the silica gel and were not able to be removed by the solvent when the
mobile phase was finally added to the toluene. This means that the molecules had more time
to adhere to the silica gel before any intermolecular forces could form between them and the
molecules in the solvent. The components would have separated more cleanly if this had not
occurred since the molecules would have been more drawn to the solvent. If I had added the
silica gel to the toluene as soon as I added the plant pigments, this could have been better.

Paper Chromatography (Plant Pigments)

This chromatography was carried out in the same way as the TLC paper pigments
chromatograph but instead used paper chromatography (partition) the same as the amino acid
chromatograph. We drew a line in pencil and smeared the plant pigments along it, then left it
in toluene until all the components were separated – again, toluene was used because it is
non-polar, meaning it will attract to the non-polar plant pigments.
We then drew on the solvent front and left it to dry. This chromatograph was not conclusive
at all and we did not calculate the Rf values because it simply did not work; and it was not
supposed to work. Toluene is a solvent that is only effective for adsorption chromatography;
it is not suitable for partition chromatography, which is why it did not function in the mobile
phase. We were unable to get Rf values because the particles blended so they were unable to
separate from the stationary phase. We would have been required to use a different solvent—
one that was suitable for partition chromatography—in order for this chromatograph to work
and for the components to adhere to and visibly separate from one another.

Plant Pigment Chromatographs

(Both TLC and Paper)

In summary, I think the TLC plant pigment chromatograph and amino acid
chromatograph went quite well, but I made a few amateur mistakes that would
be simple to correct if I were to do the experiment over. However, because we
used the incorrect mobile phase with respect to the stationary phase, the Plant
Pigment paper chromatograph was entirely incorrect; if I were to replicate this, I
would need to select a different mobile phase, at which point it would function.