Chromatography Lab Report
Chromatography Lab Report
The aim of this experiment is to separate a mixture of methylene blue and Fluorescein Sodium using
column chromatography (CC). In addition, to identify and measure the Rf values of p-nitro aniline and o-
nitro aniline and their mixture through thin layer chromatography (TLC).
The glassware to be used was rinsed with distilled water and dried. A chromatography column
was clamped to a stand, and a Stemless funnel was placed on top of it. Alumina was poured
through the funnel until the level of Alumina in the column reached 5 cm approximately. The
column was gently tapped in order to linearize the layer of alumina. Then, about 1 cm of
chromatography sand was added over the column using a spatula, and the column was tapped
again for the same purpose.
Then, the column was rinsed with ethanol allowing it to flow gently along the walls of the
column, until all the content is wet. An Erlenmeyer flask was placed under the column in order
to receive the first drop of ethanol. The stopcock was then closed.
In another Erlenmeyer flask, few ml of a dye mixture of methylene blue and fluorescein were
added using a pipette. Using a plastic dropper, about 3 drops of the dye was added over the
column. Few ml of ethanol were added slowly, and the stopcock was opened, allowing the
ethanol to elute, carrying the dyes. Ethanol was always added over certain time intervals, never
allowing the top of the column to dry.
Experiment 2: TLC
The second experiment was performed while waiting for the dyes in the first experiment to
elute. Using a pencil and a ruler, a straight line above the bottom level of a thin layer
chromatography plate (TLC plate) was drawn gently.
Using separate capillary tubes, a small drop of p-nitroaniline was placed on the left of the line,
another small drop of o-nitroaniline was placed on its right, and one last drop of a mixture of
both at the extreme right.
Then, few ml of mixture of dichloromethane and cholorform were added in a 250 ml beaker, so
that the level of solvent inside the beaker did not exceed the straight line. The beaker was
placed on the bench, and the TLC plate was put inside it. The beaker was covered with a
glasswatch, the solvent was left to rise up the plate, carrying the dyes with it. When the solvent
reached approximately the top of the paper, the paper was removed from the beaker, a using a
pencil, a mark was drawn at the level to which the solvent reached so that x travelled by the
solvent=3.1 cm, y(p-nitroaniline)=1.6 cm and y(o-nitroaniline)=2.1 cm. Then a straight line was
drawn at that mark, and the centers of each spot were located. Using the distances of migration
of the solvent and the 2 organic compounds, the rate of flow of each of the 2 compounds was
calculated.
The higher the Rf the less polar the compound is so p-nitroaniline is more polar than o-nitroaniline
Discussion:
In the first part of the experiment, column chromatography was performed using Alumina, in order to
separate the two dyes methylene blue and fluorescein sodium. The glassware were rinsed with water in
order to remove any contamination. The column was dried in order to make sure that no Alumina
powder will stick to its walls while filling it. Only about 5 cm of Alumina was added, because the longer
the alumina column is, the more time would the separation take. Thus, enough alumina was only added
to allow clear and good separation. The column was tapped gently in order to linearize the upper
surface of alumina, and ensure that the powder has been tightly packed into the column in uniform
condensed layers, leaving no air bubbles or gaps. This is important to carry out good chromatography
and ensure an easier and better separation without increasing the rate of separation of the
compounds.Alumina was used for this experiment, because it is a very polar adsorbent that acts as a
molecular sieve, separating the polar components of a mixture from the non polar ones. It is also
insoluble in water, inert with respect to the dyes used, and has a uniform composition.1 cm of sand was
added over the alumina, because when the solvent is added to the column, it won’t disturb the
stationary phase (alumina) anymore, ensuring that the alumina level remains stable and the compounds
are adsorbed at the same level. So the sand protects the stationary phase from being disturbed by the
addition of the solvent (mobile phase). The upper layer of alumina should remain uniform and linear
throughout the experiment, which is ensured by the sand, as seen in the below diagram.
The alumina in the column was wetted by flushing it with a small amount of the mobile phase ethanol.
This is called wet loading, and it helps to compact the column more. The surface of the column should
never be left to dry, and therefore, the solvent was continuously added in small amounts, until the first
drop of ethanol was collected. Then, the stopcock was closed to stop eluting ethanol so that the upper
surface does not dry out. If the surface was left to dry, cracks would form in the column of alumina,
affecting the separation process. That is why it was important to never leave the surface dry.Only 3
drops approximately of dye was added over the column, in order to observe the separation at a shorter
duration of time. Methylene blue migrated first in the column using ethanol as the mobile phase. This is
due to the difference in polarity between methylene blue and fluorescein sodium. Knowing that alumina
is a very polar adsorbent, it is able to interact with substances of similar polarity, by certain
intermolecular interactions including hydrogen bonding, dipole-dipole interactions…Methylene blue
being the first to migrate is therefore concluded to be less polar than the other dye, fluorescein sodium,
which was retained on the surface of the column when ethanol was used. The first drop of faint blue dye
indicated that the dye has begun eluting, and therefore, a new E. flask was placed in order to collect this
dye separately from the ethanol. When the last drop of blue was collected, the ethanol on the surface of
the column was left to drain down the column, and a more polar solvent was used, which is acetic acid.
The flask containing the methylene blue was placed aside to avoid its mixing with the second
solvent.The initial flask was placed in order to collect the solvent until the first drop of fluorescein
sodium has been collected. Since it was the second dye to be collected (retained longer in the column),
it can be concluded that methyl blue was able to form intermolecular interactions with the adsorbent,
and consequently, it is a more polar dye. The presence of the negative oxygen in the structure of
fluorescein could suggest the reason why it is more polar and able to adsorb to the polar alumina. The
structure of methylene blue does not contain any polarized oxygen, and is therefore incapable of
attaching to alumina, which might explain why it is less polar than fluorescein sodium.
H3 C
CH3 Cl-
S+ N CH3
N
H3C
N Methylene
Blue
ONa
O
NaO O
Fluorescein sodium
The second experiment was performed while the dyes were eluting for the first experiment in order to
save time. A pencil was used to draw the line on the TLC plate, because if a pen was used, the dye of the
pen would migrate along with the solvent on the plate, which results in losing track of where the spots
were placed initially, and as well smudge the whole plate. The line was drawn gently in order to avoid
cracking the thin layer of silica on the plate. A capillary tube was used to place the drops on the line, in
order to observe how far each drop would migrate, starting from the same position. It is also important
to use capillary tubes, because the spot should be small, otherwise, a smear would form upon placing
the plate in the solvent. Three separate capillary tubes were used to avoid contaminating the solutions
of the compounds p-nitroaniline and o-nitroaniline and the mixture. The mobile phase chosen was
dichloromethane because it is polar enough to separate the two compounds clearly. The last spot
containing both compounds was used in order to compare the location of each spot after migration,
with the spots of the pure compounds. In addition, it is inert with the compounds used and will not react
with them. The level of the solvent in the beaker was below the line mark drawn on the plate, in order
to avoid dipping the spits directly into the solvent and losing them. The beaker was placed on the bench
and left to settle; then the plate was placed in it, to prevent the fluctuation of the solvent and its
uniform migration in a straight line up the plate by capillary motion. The beaker was covered with a glass
watch in order to avoid the loss of the vapors of dichloromethane into the surrounding air, which is a
very volatile solvent. It is important to make sure that the atmosphere in the beaker in saturated with
the vapors of the solvent, which aids separation. The plate was removed from the beaker before the
solvent reached approximately from its upper end, in order to mark the migration distance of the
solvent x=3.1 cm. A pencil mark was placed immediately on the paper because dichloromethane is very
volatile, and it would evaporate off the plate within seconds leaving no marks. Since silica on the plate is
polar, it forms intermolecular interactions with the compounds of similar polarity, and therefore the less
polar compound would migrate faster. Since o-nitroaniline migrated higher than p-nitroaniline, it was
concluded that p-nitro is more polar than o-nitro. (yo=2.1 cm> yp=1.6 cm) (Rf of o=0.67> Rf of p= 0.52).
The higher the Rf the less polar so p-nitroaniline is more polar.The spot of the mixture showed 2
different spots indicating that an impure mixture is placed on the TLC because an impure mixture results
in more than one spot unlike a pure one which only results in a single spot. The adsorbent was silica
which is polar, so polar compounds will stick more to it and the less polar ones will leave more quickly.