Tips and tricks for the lab: Column Packing
Column chromatography is a
commonly used purification technique in labs across the world.
Done
right it can simply and quickly isolate desired compounds from a mixture. But
like many aspects of practical chemistry, the quick and efficient setting up
and running of a column is something that can take years to master. Here we
present some of the tips and tricks of the trade to help you set up the perfect
column.In a typical column (Fig. 1), the stationary phase, a solid adsorbent
normally silica gel (SiO2) or alumina (Al2O3),
is placed in a vertical glass column. The mobile phase, a liquid, is added to
the top of the column and flows down through the column by either gravity or
external pressure (flash chromatography). Separation of compounds is achieved
through the varying absorption on and interaction between the stationary and
mobile phasesThe quality of the separation depends on a variety of factors not
least of which is the absence of air bubbles in stationary phase.
Choice of Silica
or Alumina for the Stationary Phase.
Sillica
and alumina are both polar adsorbents so the more polar components in the
mixture to be separated are retained more strongly on the stationary phase and are therefore eluted
from the column last. Silica is recommended for most compounds, but as it is
slightly acidic, it preferentially retains basic compounds. Alumina is slightly
basic, so will retain acidic compounds
more strongly. It is good for separation of components that are weakly or
moderately polar and the purification of amines.Silica and alumina are both
polar adsorbents so the more polar components in the mixture to be separated
are retained more strongly on the stationary phase and are therefore eluted
from the column last. Silica is recommended for most compounds, but as it is
slightly acidic.
it preferentially retains basic compounds.
Alumina is slightly basic, so will retain acidic compounds more strongly. It is
good for separation of components that are weakly or moderately polar and the
purification of amines.Absorbent particle size affects how solvent flows
through the column. Silica or alumina are both available in a variety of
sizesThe size is given by the mesh value which refers to the number of holes in
the mesh that is used to sieve the absorbent. Thus higher meshvalues such as
"silica gel 230–400" have more holes per unit area and correspondingly
smaller particles than "silica gel 60".
Typically,
70–230 silica gel is used for gravity columns and 230–400 mesh for flash
columns.Alumina is available in types I, II, and III. This refers to the water
content of the alumina, with I having the least water and III the most. A lower
water content means there are more polar sites in the alumina free to bind
organic compounds, and polar compounds will remain on the column longer.
Alumina of activity II or III, 150 mesh, is most commonly employed.The
techniques for packing a column described below use silica as the stationary
phase, but are equally suitable for use with alumina
Preparing the Column.
Some
columns have glass frits to prevent loss of the stationary phase out the
bottom; others do not and will need to be plugged with either glass wool or
cotton wool. Which you use is personal preference. Positioning the cotton or
glass wool can be awkward at first, but glass frits are harder to clean and may
be a source of impurities, such as silica leaking through the frit into the
collected fractions.This can be prevented by adding a layer of sand between the
frit and the silica .The porosity of frits can also vary.This means that the
rate of solvent flow can be different for different columns .Very porous frits
will leak more silica ,but less porous frits have slower flow rates slow
sometimes too and can lead to
pressure build up in flash chromatography.
Fritted column
1. Find a clean empty column of suitable
size
2. Clamp the column securely and close
the tap or stopcock.
3. Add a layer of sand (approx 0.5cm,
optional)
Non fritted
column.
The ball of
cotton or glass wool should be large enough to plug the bottom of the column but
not so large and densely packed that it restricts solvent flow A piece the size
of the tip of your little finger should be suitable for most columns.
1.
Position the cotton or glass wool ball
securely in the narrowest part of the column using a long glass rod or other
suitable device.
2.
Clamp the column securely and close
the tap or stopcock
3.
Add a layer of sand until it reaches
the main body of the column This will give the stationary phase an even base
and prevent concentration and streaking of the bands as they come off the
column and are collected.
Filling the column.
There are
several methods for filling columns. You may find one method easier or quicker
than the others and always fill a column that way, or you may find that
different size columns require different methods. All methods have their pros
and cons and you may need to try all three to find the one that you prefer.
Dry Pack Method 1.
You will need
Funnel suitable for dry solids
Something to tap the column with
Silica or alumina
Method
1. Fill
the column with solvent, allowing some to run through the sand and cotton wool
to remove air bubbles
2. Place
a dry funnel in the top and gently pour the silica or alumina into the solvent.
Allow the solvent to drain to prevent overflowing
3. Let
the stationary phase settle and gently tap the column so that the silica or
alumina will pack tightly into the column
4. Drain
the solvent until the solvent level is just even with the surface of the phase
Dry-Pack Method 2.
Method
1.
Add
silica
gel to the column and apply house vacuum by attaching the vacuum tubing to the
bottom of the column This will compress the silica gel and keep it compressed
for the next steps. Packing can be improved by tapping the column.
2.
With the vacuum still applied, pour in
the solvent
3.
Allow the solvent to flow though the
column until it is almost at the bottom At this point, close the stopcock and
remove the vacuum line
4.
Allow 5–6 columns worth of solvent to
flow through the column to ensure complete packing.
5.
Drain the solvent until the solvent
level is just even with the surface of the stationary phase
Slurry Method:
Method
1.
Fill the column about one third with solvent
2.
In a separate flask or beaker, measure solvent
approximately one and a half times the volume of silica.
3.
In a beaker, measure out the required amount of
silica or alumina.
4.
Add the silica to the solvent, a little at a time,
while swirling. Use a Pasteur pipette or glass rod to mix the slurry.
5.
Pour pipette some of the slurry into the column. or
Allow the solvent to drain to prevent overflowing
6.
Tap the column gently to encourage bubbles to rise
and the silica to settle
7.
Continue to transfer the slurry to the column until all
the silica or alumina is added.
8.
Rinse the inside of the column by pipetting solvent
down the inside edge.
9.
Drain the solvent until the solvent level is just
even with the surface of the stationary phase
Emptying the
column:
Once
you have your products isolated, all that remains is to empty and clean the
column ready for next time. To speed up the process, elute all of the solvent
using compressed air and allow air to flow through the column for approximately
2 h. This will give dry, free-flowing silica that is easy to pour into the
silica waste container. Alternatively, elute all the solvent and secure the
column upside down over a large beaker and allow to dry overnight in a fumehoo
Cleaning the column by rinsing with water and acetone is usually sufficient.
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