C33 chromatography

• chromatography is an analytical technique used to separate and identify component molecules of a mixture
• there are many types of chromatography but all have a mobile phase and a stationary phase
• the mobile phase carries the soluble components of the mixture with it, the more soluble the component is, the faster it will move
• the stationary phase holds back the components in the mixture that are attracted to it, the more affinity a component has, the slower it will move
• an Rf value can be calculated using the equation:

  Rf = (distance moved by spot)/(distance moved by solvent)
• Rf values are used to identify the different components in a mixture
• thin-layer chromatography uses a plate coated with a thin layer of solid, silica gel or alumina, the solvent runs up the plate, then either UV light is shone on the plate or a developing agent is added to make the spots visible
• for amino acids, the developing agent is ninhydrin
• column chromatography uses a solid powder, silica or alumium oxide, packed into a column, the solvent is added to the top and runs down, the components arrive at the bottom separately so can be collected separately
• gas chromatography uses a column packed with solid powder coated with oil, and an unreactive gas as the mobile phase, the sample is injected and carried along by the gas, components separate as they have different affinities for the oil and the gas, so they arrive at different times
• retention time is the time taken for the component to leave the column after being injected in gas chromatography
• GCMS is gas chromatography-mass spectrometry
• GCMS involves a mass spectrometer as a detector for a gas chromatography system
• in GCMS, as each component of a mixture comes out of the column, the retention time is recorded and it is fed into the mass spectrometer where it can be identified by its fragmentation pattern or by its mass
• a suitable reagent for hydrolysis of a protein would be concentrated HCl or concentrated H2SO4, or a strong alkali
• amino acids have different polarities so have different solubilities in different solvents, and different retention for different stationary phases