Column Chromatography• Column Chromatography is another common and useful separation
technique in organic
• chemistry. This separation method involves the same principles as
TLC, but can be applied to separate larger quantities than TLC.
Column chromatography can be used on both a large and small scale.
The applications of this technique are wide reaching and cross many
disciplines including biology, biochemistry, microbiology and
medicine. Many common antibiotics are purified by column
• Column chromatography allows us to separate and collect the compounds
• In this experiment, Column Chromatography (abbreviated CC) will be used
to separate the mixture of ferrocene and acetylferrocene and TLC will be
used to monitor the effectiveness of this separation.
• Choosing a Stationary Phase
• As with TLC, alumina and silica are the two most popular stationary phases
in column chromatography.
• The Column Chromatography more polar sample will be retained on the
stationary phase longer.
• Thus the least polar compound will elute from the column first, followed by
each compound in order of increasing polarity.
• Choosing Solvents
• Solvent systems for use as mobile phases in CC can be determined from previous TLC
experiments, the literature, or experimentally.
• Normally, a separation will begin by using nonpolar or low polarity solvent, allowing the
compounds to adsorb to the stationary phase, then slowly switching the polarity of the solvent to
desorb the compounds and allow them to travel with the mobile phase.
• The polarity of the solvents should be changed gradually.
• On a macroscale, the mixing of two solvents can create heat and crack the column leading to a
• Some typical solvent combinations are ligroin-dichloromethane, hexane-ethyl acetate and
• Often an experimentally determined ratio of these solvents can sufficiently separate most
• Solvents such as methanol and water are normally not used because they can destroy the
integrity of the stationary phase by dissolving some of the silica gel.
• Columns can be as thin as a pencil to a diameter of several feet in industrial
• They can separate milligram to kilogram quantities of materials. In this
experiment, we will be separating a mixture of approximately 50 mg, so a
small column can be used.
• It is essential to have several clean tared Erlenmeyer flasks, reaction tubes,
beakers, test tubes or vials available to collect the solvent and compounds
as they elute.
• Once you have the general set-up prepared, you can move on to packing
the stationary phase in the column.
• 1. Using gloves, obtain a TLC plate.
• 2. Use a ruler and draw a straight line across the TLC plate exactly 1.0 cm from the
bottom of the plate.
• 3. Draw three hash marks evenly spread across the straight line. Begin assembly of the
• 4. Obtain a column with a stopcock mechanism and pack the bottom of the column with
a small amount of cotton. Use a glass rod to pack the cotton at the bottom of the
• 5. Fill the bottom of the column with 1.0 cm of sand. Proceed to add 13.0 mL of silica.
Afterwards, securely attach the column to a stand.
• 6. Add another 1.0 cm of sand to the top of the silica layer.
• 7. Using a 50.0 mL beaker, obtain approximately 0.150 g of acetylferrocene and ferrocene
mixture. Add 2.0 mL of dichloromethane to the mixture, ensuring the acetylferrocene
and ferrocene mixture goes into solution.
• 8. Add 10.0 mL of hexane to the column with a collection beaker on the bottom. Add hexane until it is 1.0 cm
from the top of the column.
• 9. Using a pipette, add acetylferrocene and ferrocene mixture. mixture one drop at a time. Then proceed to
add 10.0 mL of hexane.
• 10. Wait until a yellow solution begins to drip from the bottom of the column. Once a yellow solution begins
to drip, place a small flask to collect the solution.
• 11. After all of the yellow residue has left the column, add 10.0 mL of 50:50 hexane-ethyl acetate to the
column. Once orange solution begins to drip from the bottom of the stopcock, place a second small flask for
• 12. After collecting the second solution, there should be two flasks: one orange and one yellow flask. Use a
TLC capillary to spot each solution onto the TLC plate. Place the TLC plate into the TLC jar, allowing the 30:1
mixture to travel up the TLC plate until 2.0 cm from the top of the plate.
• 13. After spotting onto the TLC plate, place the flasks on a hot plate to evaporate. Once evaporated, weigh
the column on a weighing scale. Record the weight.
• 14. Calculate % recovery of ferrocene and acetylferrocene.
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