Chromatography - Basic Operation

Hey there! We have more interesting information about chromatography! Let's see what actually takes place in a chromatographic separation.

You can skip this review paragraph. The process of a chromatographic separation takes place within a chromatography column. This column, made of glass, plastic, 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 separations.

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.

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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.

4. Detection

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).

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Merged with material by Kevin Yip