Chromatography - Basic Operation
Hey there! We have more interesting information
about chromatography! Let's see what actually takes place in a chromatographic
You can skip this review paragraph.
The process of a chromatographic separation takes place within a chromatography column. This column, made of glass or
metal, is either a packed bed or open tubular column. A packed bed column contains particles which make up the
stationary phase. Open tubular columns are lined with a thin film stationary phase. The center of the column is hollow.
The mobile phase is typically a solvent moving through the column which carries the mixture to be separated. This can
either be a liquid or a gas, depending on the type of process. The stationary phase is usually a viscous liquid coated on the
surface of solid particles which are packed into the column as discussed above, although the solid particles can also be taken
as the stationary phase. In any case, the partitioning of solutes between the stationary and mobile phases lead to the desired
Let's see a different sketch of the process:
1. Feed Injection
The feed is injected into the mobile phase. The mobile phase flows through the system
by the action of a pump (older analytical chromatorgraphy used capillary action
or gravity to move the mobile phase).
2. Separation in the Column
As the sample flows through the column, its different components will adsorb to the
stationary phase to varying degrees. Those with strong attraction to the support move more slowly than those with weak attraction. This is how the components are separated.
Click to see an animation of separation.
3. Elution from the Column
After the sample is flushed or displaced from the stationary phase, the different
components will elute from the column at different times. The components with
the least affinity for the stationary phase (the most weakly adsorbed) will elute first,
while those with the greatest affinity for the stationary phase (the most strongly adsorbed)
will elute last.
The different components are collected as they emerge from the column. A detector
analyzes the emerging stream by measuring a property which is related to
concentration and characteristic of chemical composition. For example, the refractive
index or ultra-violet absorbence is measured.
The figure below shows a simple separation by chromatography. A continuous flow
of solvent carries a solution of solutes A and B down a column. (a) As the
solvent carries the two solutes down the column, we begin to see some
separation of the solution. (b) At some later point in time, it can be seen
that solute B is moving at a much faster rate than A. (c) In (d), solute B
emerges first, while solute A finally emerges in (e). Thus, solute A has a
greater affinity for the stationary phase than solute B. By varying the pH
of the solvent or temperature of the column, the output of the column can
be significantly altered, such as the timing of when individual species emerge.
Now...lets continue on our journey through the world of chromatography! We have
information on the output from the detector (the chromatogram).
Back to starting page
Merged with material by Kevin Yip