IA #: 5
DATE: January 12, 2015
TITLE: Separation of Methylene Blue and Methyl Orange Using Column Chromatography
AIM: To show how column chromatography can be used to separate a mixture of dyes.
APPARATUS AND MATERIALS:
Clamp and stand, two small beakers, two 100mL conical flask, one glass column(1.5cm dia.),
pipette bulb, cotton, dropper, filter funnel, wash bottle, distilled water, sand, neutral alumina,
1.0mL stock solution of methylene blue and methyl orange, solvents: ethanol and sodium
hydroxide (NaOH)
METHOD:
1. The column containing the neutral alumina was already packed with the solvent mixture.
2. The column was attached to the stand as shown in diagram. The column was clamped so
the tip was just beneath the opening of conical flask.
3. A dropper was used to remove approximately 1mL of the stock solution and to distribute
it evenly over the surface bed of alumina. Efforts were made not to disturb the surface of
alumina.
4. The solution was allowed to completely diffuse into alumina. Then a height of 1.0cm of
sand was carefully added so that the bed was completely covered.
5. The eluant (ethanol) was slowly poured onto the sand without disturbing the compound
band below.
6. After the first compound was completely eluted from the column, a second flask was
placed underneath the column to collect the second compound.
7. NaOH was then added as the new eluant and the second compound collected.
DIAGRAM
OBSERVATIONS:
Ethanol moved slowly through the neutral alumina when poured in glass column. When stock
solution was distributed over surface bed of alumina it separated into two layers: green on top
and blue at base. Blue moved further down away from green layer after sand was added. A
yellow colour was observed when ethanol was added, and after a few minutes of applying
pressure using pipette bulb the blue compound completely eluted from column. When NaOH
was added to column yellow moved down and an orange colour of increasing intensity was
observed above it. After a few minutes of applying pressure using pipette bulb the yellow
compound completely eluted from column.
DISCUSSION:
Chromatography is a technique used to separate substances from one another when they are
partitioned between a mobile phase and stationary phase. The stationary phase is a substance
which is fixed in place for the chromatography procedure. The mobile phase is the phase which
moves in a definite direction. A number of chromatographic separation are used in chemical
processes; namely column, paper, thin layer and gas- liquid chromatography. In experiment,
column or elution chromatography was employed to separate the stock solution into methylene
blue and methyl orange. The stationary phase for column chromatography is neutral alumina or
silica gel and a liquid solvent is used as mobile phase.
In experiment, neutral alumina and solvents: ethanol and sodium hydroxide were used as the
stationary and mobile phases, respectively. Sand was used to cover compound mixture before
addition of eluant to prevent the mixing of the mobile and stationary phase. The column was
kept wet to prevent the formation of air spaces as components would have been able to travel
through. This would disturb separation in column. It was observed that the blue compound
(methylene blue) eluted before the yellow compound (methyl orange). Methyl orange is more
polar than methylene blue and will have a greater affinity for the more polar solvent, ethanol.
Hence, methylene blue spent a longer time in ethanol than methyl orange and was eluted only
after sodium hydroxide was added.
The blue compound is obviously more polar than the yellow one - it perhaps
even has the ability to hydrogen bond. You can tell this because the blue
compound doesn't travel through the column very quickly. That means that it
must adsorb more strongly to the silica gel or alumina than the yellow one.
The less polar yellow one spends more of its time in the solvent and therefore
washes through the column much faster.
CONCLUSION: A mixture of dyes was separated into a blue compound (methylene blue) and
a yellow compound (methyl orange) using column chromatography with neutral alumina as
stationary phase and solvents: ethanol and sodium hydroxide as mobile phase.
