Preparing a Silica Gel Chromatography Column

The following set of instructions will assist the reader in the preparation of a silica gel chromatography column. A silica
gel chromatography column is a device that relies on silica gel to separate the components of a chemical mixture; the
separation is accomplished because each component of the mixture has a different polarity. More polar compounds
will flow easily through the silica gel, while non-polar compounds will flow more slowly through the gel. Silica gel itself
in non-polar, and thus is attracted to other non-polar molecules. The attraction of the non-polar molecules to the silica
gel is what causes the non-polar components of a mixture to move slowly through the gel. Chemical separation relies
on the relative speeds at which each component travels from the top of the column to the bottom when using a silica
gel chromatography column. The length of the column will provide a separation that gives a high purity for each
component; each component while drain out of the column and have a sharp ending point. None of the liquid drained
from the bottom of the column will have both components of the mixture in it. A full separation (no product is left in the
column) of the mixture will also occur within one hour when using the column outlined in these instructions.

Required Equipment

• Glass column with a stopcock • 2 500 mL beakers

• Pipette, equipped with pipette bulb • 150 mL graduated cylinder
• Flat-necked glass funnel • 2 three-fingered clamps
• 250 mL Erlenmeyer flask • Ring stand
• One cotton ball • Metal scupula
• 1 Coat hanger, straightened • Glass stirring rod

Required Chemicals

• 8 grams of reagent-grade sand

• 200 grams of silica gel
• 150 mL of dry diethyl ether

Warning: Diethyl ether is extremely flammable; do not use it near open flame

• 150 mL of cyclohexane

Warning: cyclohexane is extremely flammable; do not use it near open flame

• Mixture to be separated

Procedure
Warning: Due to the flammability of the solvents (diethyl ether and cyclohexane), the preparation
of the chromatography column should be performed in the laboratory’s fume hood. Failure to
perform the procedure in the fume hood could result in injury and equipment damage.

All glassware should be clean and dry before it is used. Failure to clean and dry glassware will result in a poor
separation or unwanted side reactions, ruining any previously prepared products.

1. Pour the 150 mL of diethyl ether and the 150 mL of cyclohexane into the 500 mL beaker. Use the glass
stirring rod, stirring the solution for two minutes. This solution will serve as your eluent and will be
needed for the separation of your compound.
2. Place the two three-fingered clamps on the ring
stand. Put the glass column into the three-fingered
clamps - placing one clamp just above the column’s
stopcock and the other clamp roughly three inches
from the top of the column. See Figure 1 for a
diagram of the setup.

Caution:
Caution: DoDonotnot over-tighten
overtighten thethe three- ered
three-fing
fingered clamps, as it could crack or break
clamps, as they could crack or break the glass
the glass column.
column.

3. Close the stopcock. Tear the cotton ball in two, and

place one of the halves into the glass column. Use
the straightened coat hanger to gently push the
cotton to the bottom of the glass column.

Caution:
Caution: DoDonotnot
trytry
to to compact
compact thethe cotton.
cotton. Simply
Simply
push push the
the cotton cotton
to the plugoftothe
bottom thecolumn,
bottom oasf the
the column.
cotton Compacting
plug is only needed to theprevent
cotton sand
plug may
from Figure 1
clog the column, preventing it from working. Clamps on the Glass Column

3. Measure 4 grams of sand and pour it into the

column using the scupula. Gently shake the
column so that the sand forms a flat surface.
See Figure 2.

4. Pour the 200 grams of silica gel into the empty

500 mL beaker. Pour roughly half of the
mixture prepared in step 1 into the 500 mL
beaker that the silica gel is in. Stir the solution
gently.

5. Set the flat-necked glass funnel on top of the

column. Pour the solution prepared in step 4
through the funnel so that it flows into the
column and comes to rest on top of the layer of
sand. See Figure 2.

Caution:
Caution: Pour
Pour thethe solution
solution in in slowlyand
slowly and
carefullyand
carefully, so that the disrupt
do not sand layer is notlayer. If
the sand
thedisturbed.
silica gel Iftouches
the silica
thegel touches
cotton thepoint,
at any
thecotton
columnat any
maypoint, the column
not function may not
correctly.
function correctly.

6. Leave the flat-necked funnel on top of the column and

allow the silica gel layer to settle for five minutes.

7. Pour roughly 5 mL of the remaining diethyl-

ether/cyclohexane mixture prepared in step 1 into the
funnel.

Repeat this step until all of the silica gel is cleaned

from the walls of the column. The silica gel must form
one layer, and not remain on the walls of the column
for proper product separation.
 8. Remove the funnel from the top of the column
and use the scupula to pour the remaining sand
onto the silica gel layer. The liquid sitting
on top of the silica gel layer will level the sand
without shaking or stirring. See Figure 3.

9. Place the 500 mL beaker containing the remaining

ether-cyclohexane mixture under the spout of the
column. Open the stopcock, allowing the liquid in the
column to flow out until the liquid’s level is slightly
above the top of the sand layer. This will eliminate air
bubbles from the column. See Figure 4 for an image
of the column after draining the excess solution.

Close the stopcock when finished.

Caution: Do not let the liquid level ever get below the
Caution: Do not let the liquid level get below the
top of the silica gel layer. Allowing the liquid layer to
top of the silica gel layer. Allowing the liquid layer
get below the silica will ruin the separation power of
to get below the top of the silica gel will ruin the
your column. If the liquid layer gets below that top
separation power of the column. If the liquid
of the silica gel, the column should not be use.
layer gets below the top of the silica gel, you
You should start the column preparation over
will need to discard the current column and
should the liquid layer get below the silica gel
assemble a new one.
layer.

10. With the stopcock closed, transfer your sample to

the pipette. Pipette the solution into the column. Allow
it to run down the side of the column, so that it does
not perturb the sand layer. See Figure 5 for an
illustration of proper pouring technique.
Caution: Be sure to let the sample run down the
Caution: Be sure to let the sample run down the side
side of the column. If the sample gets below the
of the column. If the sample gets below the sand,
sand, separation of your sample mixture will be
separation of your sample mixture will be poor. Figure 4
poor.
Column After Draining Excess Solution

11. Place the Erlenmeyer flask beneath the spout of

the column.

Figure 5
A Comparison of Proper Product Introduction Technique
Be sure not to pour the product directly onto the sand. Use the wall of the column to avoid disturbing the sand.
 You have produced a
chromatography column that
will be able to separate all of
the two-compound product
mixtures produced in the
Organic Chemistry Laboratory
at Texas Tech. You also have
the supply of eluent (the
excess liquid prepared in step
1) needed to carry out a
separation. Your column
should look like Figure 6,
opposite this paragraph.

Figure 6
Finished Chromatography Column
.

Common Column Problems and Precautions

Problem Cause Prevention

When stopcock is opened, the Cotton is packed too tightly into the Do not compact the cotton with the
column does not drain. bottom of the column. hanger. The cotton ball should still be
fluffy when pushed to the bottom of
the column.
Eluent drains out of the column, but Silica gel is bonding to the product Do not allow the liquid layer to go
none of the product flows into the due to part of the gel having dried. below the top of the silica gel layer.
collection flask. When the silica gel dries out, it will
begin to bond compounds to itself.
The column works, but product 1. The air bubbles were not removed 1. Do not forget to drain the excess
separation is poor. The product from the column, causing the product eluent from above the sand layer
components are coming out as a to be moved through the column before introducing your product.
mixture, instead of coming out more slowly.
separately. 2. Do not drop your product into the
2. The product penetrated the sand middle of the column, but pour the
as it was being pipetted into the product down the side of the column.
column.