In column chromatography, the stationary phase, a solid
adsorbent, is placed in a vertical glass (usually) column and the mobile phase,
a liquid, is added to the top and flows down through the column (by either
gravity or external pressure). Column chromatography is generally used as a
purification technique: it isolates desired compounds from a mixture.
The mixture to be analyzed by column chromatrography is
applied to the top of the column. The liquid solvent (the eluent) is passed
through the column by gravity or by the application of air pressure. An
equilibrium is established between the solute adsorbed on the adsorbent and the
eluting solvent flowing down through the column. Because the different
components in the mixture have different interactions with the stationary and
mobile phases, they will be carried along with the mobile phase to varying
degrees and a separation will be achieved. The individual components, or
elutants, are collected as the solvent drips from the bottom of the column.
Column chromatography is separated into two categories,
depending on how the solvent flows down the column. If the solvent is allowed to
flow down the column by gravity, or percolation, it is called gravity column
chromatography. If the solvent is forced down the column by positive air
pressure, it is called flash chromatography, a "state of the art" method
currently used in organic chemistry research laboratories.
Silica gel (SiO2) and alumina (Al2O3) are
two adsorbents commonly used by the organic chemist for column chromatography.
These adsorbents are sold in different mesh sizes, as indicated by a number on
the bottle label: “silica gel 60” or “silica gel 230-400” are examples. This
number refers to the mesh of the sieve used to size the silica, specifically,
the number of holes in the mesh or sieve through which the crude silica particle
mixture is passed in the manufacturing process. If there are more holes per unit
area, those holes are smaller, thus allowing only smaller silica particles go
through the sieve. The relationship is: the larger the mesh size, the smaller
the adsorbent particles.
Adsorbent particle size affects how the solvent flows
through the column. Smaller particles (higher mesh values) are used for flash
chromatography, larger particles (lower mesh values) are used for gravity
chromatography. For example, 70–230 silica gel is used for gravity columns and
230–400 mesh for flash columns.
Results were less than acceptable when
large 60-200 mesh material was used, but remarkably improved when a 200-400
mesh material was in the column. Equally important: particle sizes less than 40
microns offered no significant improvement in resolution in this system.
The polarity of the solvent which is passed through the
column affects the relative rates at which compounds move through the column.
Polar solvents can more effectively compete with the polar molecules of a
mixture for the polar sites on the adsorbent surface and will also better
solvate the polar constituents. Consequently, a highly polar solvent will move
even highly polar molecules rapidly through the column. If a solvent is too
polar, movement becomes too rapid, and little or no separation of the components
of a mixture will result. If a solvent is not polar enough, no compounds will
elute from the column. Proper choice of an eluting solvent is thus crucial to
the successful application of column chromatography as a separation technique.
Often a series of increasingly polar solvent systems are
used to elute a column. A non-polar solvent is first used to elute a less-polar
compound. Once the less-polar compound is off the column, a more-polar solvent
is added to the column to elute the more-polar compound.
Column Chromatography Procedure
Packing a (silica gel) column:
- Use a piece of wire to add a plug of cotton to the bottom of the
column. There should be just enough cotton that the sand and silica will not
fall out of the column.
- Clamp the column to a ring stand and add enough sand to fill the
curved portion of the column.
- Place a pinch clamp on the tubing, then fill the column 1/4 to 1/3
full with the initial eluent.
- Prepare a slurry of silica in the
initial eluent by pouring dry silica
into a beaker of eluent. (Add a volume of silica gel, such as 20 mL, to
approximately double the volume of eluent, 40 mL.) CAUTION: keep the dry
your hood and be careful not to inhale the lightweight substance.
- Quickly but carefully pour the slurry into the column. Stir
and pour immediately to maximize the amount of silica that goes into the
column instead of remaining behind in the beaker. You may find a clean
spatula or glass rod helpful in transferring the silica.
- Remove the pinch clamp to allow solvent to drip into a clean
flask. Tap on the side of the column with a rubber stopper or tubing to
help the silica settle uniformly.
- Use a Pasteur pipet to rinse any silica that is sticking to the sides
of the column. Allow the silica to settle while eluent continues to drip
into the flask.
- Once the silica has settled, carefully add sand to the top of the
column. Sand is heavier than silica. If the silica has not settled, the
sand may sink into the silica instead of forming a layer on top of it.
(You may need to rinse down sand that sticks to the side of the column.
Loading a sample onto the column:
- Drain eluent from the column until no solvent remains above
the surface of the sand.
- Using a long Pasteur pipet, carefully add your sample to the column.
- Drain eluent from the column until no sample remains above the surface
of the sand.
- Use ~ 1 mL of eluent to rinse your container and pipet. Add this
milliliter of sample to the sand. Drain eluent from the column until no
liquid remains above the surface of the sand.
- Repeat step 12 two or three times to completely transfer your sample
onto the silica gel. If you do not do and repeat step 12, your sample
will remain in the sand instead of on the silica. Sample remaining in the
sand will dissolve in the eluent that you add in step 14, ruining the
possibility of good separation of components.
Eluting the sample:
- Once you have rinsed your sample onto the silica, carefully
add eluent to the top of the column. To avoid disturbing the top of the
column, it's a good idea to carefully pipet an inch or two of solvent onto
the column instead of pouring solvent directly onto the sand.
- Add more eluent as necessary. The eluent collected prior to the
elution of sample can be recycled. The composition of the eluent can be
changed as the column progresses. If the eluent composition is to be
changed, ALWAYS start with least polar solvent/mixture and change to the
more polar solvent/mixture.
Analyzing the fractions:
- Analyze the fractions by thin-layer chromatography to
determine a) if the fraction contains more than one component and b) if
fractions can be combined without affecting the purity of those fractions.
- The success of your separation will be dependant on how well you pack and
load the column. It is important to have level sand and silica. It is also
important to carefully and evenly add your sample to the packed column.
- Do not allow the silica to dry out as the column progresses. Cracks will
form within the silica column if it dries, and compounds can fall down the
cracks instead of partitioning between mobile and stationary phases.
- Compounds pass through sand quickly and do not stick to it. Sand is used
at the bottom of the column to help ensure a level silica gel line. The bottom
of the column is typically cone shaped. If no sand were present at the bottom
of the column, molecules traveling down the center of the column would
encounter less silica gel than molecules traveling down the edge, closer to
the glass. As a result, a particular component would elute as a broader band
which is undesirable.
- Sand is used at the top of the column to aid even loading of the sample.
Sample diffuses evenly through the sand. Once the pinch clamp is removed from
the bottom of the column, sample loads evenly onto the silica. Without sand,
the sample would be added directly to the silica and would stick wherever it
is added, not evenly across the surface of the silica.