SBA #
Date: January 17, 2019
Title: Separation of a mixture of methylene blue and methyl orange using column chromatography
Aim: To show how column chromatography can be used to separate a mixture of dyes
Materials/Apparatus:
Stock solution of methylene blue and methyl orange mixture
Two conical flasks
Glass column
Cotton
Neutral alumina
Clamp and stand
Ethanol
Water
Set up of apparatus
Theory
Chromatography is a technique used to separate the components of a mixture. This method works by
dividing the mixture between two phases known as the stationary and mobile phase. This is known as
partitioning. The mixture to be separated is introduced into the stationary phase, which can be a solid such
as silica (silicon (iv) oxide) or alumina (aluminum oxide) which stays still and tends to hold back the
components of the mixture which are attracted to it. The mobile phase which is a liquid (solvent or
solvent mixture), is then allowed to move over the stationary phase. This carries the components of the
mixture that are attracted to the stationary phase in the direction of flow resulting in the separation of
these components. Partition depends on the different solubilities of the components in the mobile phase
and the different absorption (temporary attraction of molecules of liquid to a solid surface) of the
components with the stationary phase. Components with greater with greater solubilities will dissolve in
and move readily along the mobile phase during chromatography. Components with stronger absorption
forces will be held on the stationary phase and not move along with the mobile phase readily.
There are 4 types of chromatography. These include: Column chromatography, Thin-layer
chromatography, Paper chromatography and Gas-liquid chromatography. Column chromatography is a
technique used to separate a mixture of chemical substances into its individual compounds. The apparatus
for carrying out this technique is a simply a glass or plastic tube called a column which is usually held
vertically by a retort stand. Within this tube there is solid of resin, silica or alumina which forms the
stationary phase, a mineral wool or sintered glass plug at the bottom, a solvent (mobile phase) such as
alcohol or water, sand and the mixture to be separated, which is added to the top of the column and
allowed to soak into the stationary phase. The solvent (mobile phase) is then continuously added to the
top of the column. The solvent moves through the stationary phase and carry the components in the
direction of flow resulting in separation.
Method
The glass column containing the neutral alumina was packed with the solvent mixture. The column was
attached to the stand so that the tip is just beneath the opening of the conical flask.
A dropper was used to remove and distribute approximately 1ml of the stock solution and distribute it
over the surface bed of alumina. It was ensured that the surface of the alumina was not disturbed.
The solution was allowed to completely diffuse into the alumina then sand was added carefully to a
height of 1cm so that the bed is completely covered.
5ml of the eluent (ethanol) was poured slowly onto the sand without disturbing the compound band
below. As soon as the first compound was completely eluted from the column, a second flask was placed
underneath the column to collect the second compound. Water was then added as new eluant and the
second compound was collected. The third compound was collected in another conical flask.
Observations
It was observed that when the eluent was added to the column, it had passed through the silica causing the
separation of the green colored sample mixture into 3 different component colors. The first color obtained
was blue followed by green followed by yellow-orange.
FIGURE 1 SHOWING THE OBSERVATIONS OF THE THREE FRACTIONS THAT PARTIONED
USING COLUMN CHROMOTOGRAPHY
FIGURE 1.1 SHOWING THE SECOND COMPONENT SEPARTED FROM A MIXTURE OF DYES
USING COLUMN CHROMATOGRAPHY
Discussion
In the experiment done, a piece of cotton was placed at the bottom of the flask to prevent alumina from
running out during chromatography. The alumina (absorbent) was then packed into the column on top of
the cotton. The green stock solution was then evenly distributed to the surface bed of the alumina by
adding the solution to the side of the glass column so that the top of the stationary phase(alumina) is not
disturbed. The stock solution was allowed to completely diffuse into the alumina and 1 cm of sand was
added to the bed so it is completely covered. The eluent which is a solvent used for washing a compound
through the column (ethanol), was then slowly poured onto the sand without disturbing the compound
band beneath. The tap at the bottom of the column was opened causing the downward movement of
eluent through the column. However instead of relying on gravity a pipette filler was used to speed up the
motion of the eluent through the column and increase the speed at which the components of the mixture
were separated. The solvent was added continuously to the column until all the components were obtained
as well as prevent the stationary phase (alumina) from becoming dry which can cause swelling followed
by cracking. This will cause the eluent to rush through the cracks therefore increasing the likelihood of
the mixture not separating into the components.
While the mobile phase was moving through the stationary phase it was observed that the sample
mixture had separated into three colored compounds. The first compound collected was methylene blue,
had a blue color. The second compound collected had a green color and the final compound obtained was
methyl orange, had an orange color.
Since the factor which determines what component present in the mixture would separate first was the
polarity of the compounds. According to the polarity order of these compounds, orange>green>blue, it
can be said that the methylene blue was the least polar compound as it had travelled the quickest through
the column since it spends more time in the mobile phase than the stationary phase. Therefore, it is
quickly washed through the column and was the first fraction collected. The green compound had a
greater polarity than methylene blue but a lesser polarity than methyl orange therefore it was collected as
the second fraction. The methyl orange was the most polar compound. Therefore, it was more strongly
absorb to the alumina than the other compounds, thus had the tendency to move with the mobile phase
therefore it travelled the slowest through the column and was the last compound that was obtained.
After collection of the first two compounds, the speed at which methyl orange was obtained could have
been increased by adding a more polar solvent to the column. The more polar compound works in two
ways to allow this to happen. These include:
The more polar solvent will compete for space on the alumina with the methyl orange. Any space
temporarily occupied by solvent molecules on the surface of the stationary phase is not available
for the methyl orange molecules to stick and this will tend to keep them moving along in the
solvent
There will be greater attraction between the polar solvent molecules and the polar methyl orange
molecules. This will tend to attract any methyl orange molecules sticking to the stationary phase
back into the solution.
Conclusion
It can be concluded that column chromatography can be used to separate the components of a mixture as
was seen from the partitioning of the methyl orange and methylene blue from a sample mixture
