1 Chromatography

Chromatography is an analytical technique that separates components in a mixture between a mobile phase and a stationary phase. The mobile phase may be a liquid or a gas. The stationary phase may be a solid (as in thinlayer chromatography, TLC) or either a liquid or solid on a solid support (as in gas chromatography, GC) A solid stationary phase separates by adsorption, A liquid stationary phase separates by relative solubility If the stationary phase was polar and the moving phase was non- polar e.g. Hexane. Then non- polar compounds would pass through the column more quickly than polar compounds as they would have a greater solubility in the non-polar moving phase. (Think about intermolecular forces) Separation by column chromatography depends on the balance between solubility in the moving phase and retention in the stationary phase. In gas-liquid chromatography GC the mobile phase is a inert gas such as nitrogen, helium, argon. The Stationary phase is a liquid on an inert solid. Types of chromatography include: • thin-layer chromatography (TLC) – a plate is coated with a solid and a solvent moves up the plate • column chromatography (CC) – a column is packed with a solid and a solvent moves down the column • gas chromatography (GC) – a column is packed with a solid or with a solid coated by a liquid, and a gas is passed through the column under pressure at high temperature. Retention times and Rf values are used to identify different substances. Method: Thin-layer chromatography a) Wearing gloves, draw a pencil line 1 cm above the bottom of a TLC plate and mark spots for each sample, equally spaced along line. b) Use a capillary tube to add a tiny drop of each solution to a different spot and allow the plate to air dry. c) Add solvent to a chamber or large beaker with a lid so that is no more than 1cm in depth d) Place the TLC plate into the chamber, making sure that the level of the solvent is below the pencil line. Replace the lid to get a tight seal. e) When the level of the solvent reaches about 1 cm from the top of the plate, remove the plate and mark the solvent level with a pencil. Allow the plate to dry in the fume cupboard. f) Place the plate under a UV lamp in order to see the spots. Draw around them lightly in pencil. g) Calculate the Rf values of the observed spots. Rf value = distance moved by amino acid distance moved by the solvent Wear plastic gloves to prevent contamination from the hands to the plate pencil line –will not dissolve in the solvent tiny drop – too big a drop will cause different spots to merge Depth of solvent– if the solvent is too deep it will dissolve the sample spots from the plate Will get more accurate results if the solvent is allowed to rise to near the top of the plate but the Rf value can be calculated if the solvent front does not reach the top of the plate lid– to prevent evaporation of toxic solvent dry in a fume cupboard as the solvent is toxic UV lamp used if the spots are colourless and not visible N Goalby chemrevise.org 2 Gas-liquid chromatography can be used to separate mixtures of volatile liquids. In gas-liquid chromatography, the mobile phase is a gas such as helium and the stationary phase is a high boiling point The time taken for a particular compound liquid absorbed onto a solid. to travel from the injection of the sample to where it leaves the column to the detector is known as its retention time. This can be used to identify a substance. Carrier gas detector display column oven Sample in Waste outlet Flow control Some compounds have similar retention times so will not be distinguished. Basic gas-liquid chromatography will tell us how many components there are in the mixture by the number of peaks. It will also tell us the abundance of each substance. The area under each peak will be proportional to the abundance of that component. It is also possible for gas-liquid chromatography machine to be connected to a mass spectrometer, IR or NMR machine, enabling all the components in a mixture to be identified. Most commonly a mass spectrometer is combined with GC to generate a mass spectra which can be analysed or compared with a spectral database by computer for positive identification of each GC component in the mixture. -MS is used in analysis, in forensics, environmental analysis, airport security and space probes. Gas-Liquid Chromatography HPLC stands for high performance liquid chromatography and it type of column chromatography commonly used in industry. HPLC: stationary phase is a solid silica HPLC: mobile phase a liquid Column chromatography (CC) Simple column chromatography • A glass tube is filled with the stationary phase usually silica or alumina in powder form to increase the surface area. • A filter or plug is used to retain the solid in the tube. Solvent is added to cover all the powder. • The mixture to be analysed is dissolved in a minimum of a solvent and added to the column. • A solvent or mixture of solvents is then run through the column. • The time for each component in the mixture to reach the end of the column is recorded (retention time)