Basics of Chromatography
In A2/A-Level Chemistry, chromatography is a technique used to separate components in a mixture and identify them by comparing their Rf/retention time value to a database.
Mobile phase – the phase that moves --> liquid or gas
Stationary phase – the phase that doesn’t move --> solid or liquid
Chromatography works on the basis that different components have different affinities for a stationary and mobile phase. The greater the interaction the component has with the stationary phase the more it is slowed down; the difference in speeds is what creates the separation.
Methods of separation:
- Adsorption --> solid or liquid stationary phase
- Relative solubility --> liquid or gas stationary phase
Adsorption – the process by which a solid holds molecules of a gas or liquid or solute as a thin film on the surface of a solid or, more rarely, a liquid.
Methods of chromatography
In A2/A-Level Chemistry, methods of chromatography can be separated into Thin Layer Chromatography (TLC) and Gas Chromatography (GC).
For TLC:
- Method of separation: Adsorption
- Mobile phase: Liquid solvent
- Stationary phase: Silica gel (SiO2) or alumina (Al2O3) supported on a sheet of glass or plastic
Method/ process:
- Draw a sample line on the TLC plate and pipette small drops of your sample(s) on this line.
- Place the plate in a tank with a small volume of solvent that should be higher than the sample line
- Cover the tank and allow the solvent time to soak up the plate and then take it out before the solvent runs off the edge
- Mark the solvent line and allow the remaining solvent to evaporate
- Analyse the plate by circling the spots and calculating the Rf value(s) – if the samples aren’t visible use a UV light
Limitations:
- Similar compounds often have similar Rf values – components hide behind each other
- Unknown compounds have no reference Rf value for comparison– the analyst needs to have a vague idea what the component is for the results to be meaningful
- Can be difficult to find (mixture of) solvent(s) that will cause all the components to separate at an optimum rate
For GC:
- Method of separation: Relative solubility
- Mobile phase: Carrier gas (inert unreactive gas, usually helium or nitrogen)
- Stationary phase: Adsorbent (thin layer of a solid or liquid, such as a long-chained alkane (l) or silicone polymers (s), coated on the inside of coiled capillary tubing in an oven)
Method/ process:
- Sample mixture is injected into the gas chromatogram and is vaporised
- Vaporised mixture is forced through the capillary column due to the pressure from the carrier gas
- Components dissolve into/ adsorb to the stationary phase and depending on the strength of their interactions determines the rate at which each component moves through the capillary column
- A detector records the retention time (how long it takes to travel through the capillary column) of each component
- The results are processed by a computer and are then displayed in a gas chromatogram
Limitations:
- Thousands of chemicals have the same retention time, peak shape and detector response, so it doesn’t positively identify most components
- Unknown compounds have no reference retention time for comparison – the analyst needs to have a vague idea what the component is for the results to be meaningful
- Not all components will be separated and detected because some components ‘hide’ behind other components in higher concentrations with the same retention time
Chromatogram
In A2/A-Level Chemistry, it is a visible record showing the results of separation of the components of a mixture by chromatography.
Rf value is unit used to compare how far a component has travelled up a TLC plate in relation to the solvent front.
Rf = distance moved by component ÷ distance moved by solvent front
Retention time
It is the time for a component to pass from the column inlet to the detector. It isIn GC the area under each peak is proportional to the amount of a compound in the sample.
That's the end~
