Chromatography

Chromatography is an analytical technique used to separate components with similar physical properties from a mixture. In chromatography, a mobile phase sweeps a mixture over a fixed stationary phase. The greater the interaction between the stationary phase and the components in the mobile phase, the slower the components move. The difference in speed between components results in the separation.

The two main types of chromatography are:

Separation Methods

seperation

Thin Layer Chromatography

Thin layer chromatography is used for separating a mixture into its components with the use of a solid stationary phase and a liquid mobile phase.

Producing the Chromatogram

A chromatogram is a visible record showing the result of the separation. Thin layer chromatography is achieved by the adsorption of the components onto the solid stationary phase. The components which bind to the adsorbent more strongly move slower up the plate.

tlc_components
The diagram above shows the movement of the separated components as the solvent rises up the plate. The method for thin layer chromatography is shown below:

  1. First a sample of the mixture is dissolved into aqueous solution. A small drop is then placed close to the bottom of the TLC plate and allowed to dry.
  2. A horizontal pencil line should be drawn to indicate the starting position of the sample. Ink should not be used as the ink would separate on the TLC plate.
  3. The TLC plate should be placed into the beaker with a shallow layer of solvent below the sample line. The beaker should then be sealed in order to saturate the space in the jar with solvent vapour. This slows down the evaporation of the solvent, reducing solvent loss.
  4. The solvent will then rise up the plate due to capillary action. Along with the solvent, the components in the sample are also swept vertically up the TLC plate. Once the solvent has nearly reached the top of the plate, the plate should be removed and allowed to dry.
  5. A pencil line should be drawn at the maximum height reached by the solvent. The generated chromatogram should show spots of the separated mixture.

Analysing the Chromatogram

Each separated component appears as a spot on the TLC plate. Sometimes the spots may be colourless, in which case a method should be used to locate the spots:

Once the positions of the separated components have been determined, the TLC chromatograms can be interpreted through the use of $R_{f}$ values. These indicate how far the component has travelled up the TLC plate.

$$ R_{f} = \frac{\text{distance moved by component}}{\text{distance moved by solvent front}} $$
The value of $R_{f}$ has no units and will always be between $0$ and $1$. In the diagram below the distance moved by the solvent front is represented by $y$ while the distance moved by the component represented by $x$.

rf_values

If the chromatography was repeated, the same $R_{f}$ value would be determined. Unknown substances can be identified by comparing them to known values.

Limitations of TLC

Gas Chromatography

Gas chromatography is a technique used to separate volatile compounds. It takes place in an instrument called a gas chromatograph.

GC_equipment

Producing the Chromatogram

  1. The mixture is vaporised into the gas chromatograph. The mobile phase carries the mixture through the capillary column.
  2. As the components interact with the stationary phase they are slowed down by different amounts, resulting in the separation. If the stationary phase is solid, the components are slowed by adsorption. If the stationary phase is liquid, the greater the solubility of the gas into the liquid, the more they are slowed down.
  3. Each compound leaves the column and is detected at different times. A computer processes these results to produce a gas chromatogram.

Retention Time

The retention time is the time taken for a component to pass from the column inlet to the detector. As each compound is slowed by a different amount, each compound has a unique retention time. The graph below shows a chromatogram of alcohols and carbonyl compounds in a sample of blood.

gas_chromatogram

An everyday use of gas chromatography is in breathalysers to determine the amount of alcohol in the blood.

Limitations of Gas Chromatography

GC-MS

Gas chromatography and mass spectrometry can be combined into a powerful analytical tool for the identification of compounds. This process is called gas chromatography-mass spectrometry or GC-MS.

  1. Gas molecules are first separated through gas chromatography. The retention times can provide an initial identification of the molecule.
  2. The separated components can be directed into a mass spectrometer where a mass spectrum is produced with molecular ion peaks and fragment ion peaks.
  3. The generated mass spectrum can be analysed and compared with a spectral database. As each compound has a unique mass spectrum, mass spectrometry allows for the positive identification of each component in a mixture.

Uses of GC-MS

© Andrew Deniszczyc, 2018