Chromatography

​​In a nutshell

Mobile and stationary phases are fundamental to all types of chromatography and the affinity to these phases is what allows for components to be separated and identified. There are several different types of chromatography including thin layer, column and gas chromatography.  

Mobile and stationary phases

Chromatography is used to separate the components in mixtures. Once they are separated, they can be identified. The separation occurs as components have different affinities for the mobile and stationary phases. The stationary phase, where the molecules can not move, is a solid or a liquid on a solid support. The mobile phase, where the molecules can move, is a liquid or a gas. The lower the the affinity to a component's stationary phase, the more likely a substance will be in the mobile phase more often. The greater the affinity to the mobile phase the faster / further the distance the molecule will travel. 

$$R_f$$​ values

The $$R_f$$ value differs for each compound. It is calculated by measuring the distance moved by the mobile phase and the molecule.

​$$R_f=\ \frac{Distance\ moved\ by\ molecule}{Distance\ moved\ by\ mobile\ phase}$$​​

Different types of chromatography will have different $$R_f$$​ values for each molecule. 

Thin-layer chromatography

In thin-layer chromatography (TLC), a metal or glass plate is coated in a thin layer of silica (silicon dioxide) or alumina (aluminium oxide) and the solvent moves up the plate. As the solvent moves up the plate (this is the mobile phase), it carries the components of the mixture with it. Certain components will have a greater affinity for the solvent than others and move up the plate faster. 

The plate is then allowed to dry. Certain chemicals such as the components of dye can be seen with the naked eye. However, compounds such as amino acids are colourless. These chemical traces can be seen under UV light and the distances travelled by the compounds and the solvent can be measured. Additionally, a developing agent can be added to the plate to help see the traces to the naked eye or to help to see the stains under a UV lamp.

Column chromatography

Column chromatography is typically used to purify an organic product. A glass vertical column is packed with a powdered solid and forms a slurry and will act as the stationary phase. A solvent which contains the compound being analysed is then added to the top and is allowed to run through the slurry - this is the mobile phase. 

The different affinities will result in the compounds draining out of the columns at different rates. They can be collected as separate samples. The time taken to drain can be used to identify a compound as the retention time differs depending on the compound.

Gas chromatography

This type of chromatography is used if the mixture contains volatile liquids (liquids that evaporate easily). A thin tube is packed with a powdered solid or a solid coated with a viscous liquid, which will act as the stationary phase. High-pressure gas, typically nitrogen, is passed through the tube as the mobile phase. The analysis records the retention time for each molecule in the mixture, allowing them to be identified as each substance will have its own retention time. Additionally, the area under the peak will tell you the relative amount of substance present. 

GC-MS

This is a combination of gas chromatography and mass spectrometry. Firstly, molecules are separated according to their affinities to the stationary phase in gas chromatography. Then, the molecule is fed into the mass spectrometer to be identified. Mass spectrometry involves finding the charge-to-mass ratio of compounds. GC-MS is very fast and has accurate molecule identification.