Chromatography an analytical technique for

this is experimental technique for quantitative and qualitative analysis

1. Chromatography

2. C hromatography Chromatography is a technique to separate and identify components in a mixture There are many types of chromatography including: Paper chromatography Thin layer chromatography (TLC) Column chromatography Gas chromatography (GC) B A A+B

3. Phases Two phases are required for separation in chromatography: Stationary phase Mobile phase Stationary phase – the phase that the compound travel across that does not move, must be solid or liquid on a solid support Mobile phase – is the carrier for compounds through the stationary phase, is either liquid or gas Separation depends on the balance between solubility in the mobile phase and retention by the stationary phase

4. How it works All types of chromatography use the same basic principles: The mobile phase moves over or through the stationary phase The solubility of the substance in the mobile phase and its retention by the stationary phase determines the distance the substance moves up the plate The more the substance is soluble in the mobile phase the further up the plate it will travel The difference in solubility in the mobile phase and retention by the stationary phase, leads to separation of different substances

5. Paper chromatography Stationary phase – paper (cellulose particles) Mobile phase – water or propanone Limited number of solvents can be used with paper, limited number of compounds can be tested using this method Mainly water soluble inks

6. Beaker TLC Plate Solvent Solvent front Baseline B A A+B B A A+B Thin Layer C hromatography (TLC) At time zero After 10 minutes A plate is coated with a solid and a solvent moves up the plate Stationary phase – Absorbent material such as silica (silicon dioxide) or aluminium oxide on a glass or metal plate Mobile phase – Solvent (can be altered depending on the compound) The purity of a sample can be determined – sample is impure if more than one dot appears Watch glass lid

7. Seeing colourless chemicals Both coloured and colourless chemicals can be separated by TLC Colourless chemicals will not be seen There needs to be a way of making the colourless dots visible: UV light Iodine vapour UV light – TLC plate contains fluorescent dye, under a UV light the spots where the chemicals are will up as dark patches Iodine vapour – The TLC plate is placed in a sealed jar with iodine crystals. Iodine vapour sticks to the chemicals on the plate Spots turn brown/purple

8. Running a TLC Base line is drawn onto TLC plate. Sample is spotted onto TLC plate. TLC plate is placed into solvent. Lid is placed on top. TLC plate is taken out of jar. Solvent front drawn on. Sample is colourless so placed under UV. Spots are circled. Rf can be calculated.

9. Retention factor Retention factor ( R f ) - distance travelled by the centre of a spot to the distance travelled by the solvent front. Can be used to find out what the chemical is. R f R f value for a compound changes depending on the solvent, TLC plate and temperature. Each compound has a specific retention factor value. F or an unknown sample the R f value can be compared to a database of R f values to find the compound. If you suspect your sample contains a specific compound you can spot that compound alongside the mixture to see if one of the spots matches it.   Solvent front Baseline

10. Column Chromatography Column chromatography is mainly used to purify an organic product Stationary phase – packed column with a slurry of absorbent material such as silica or aluminium oxide Mobile phase – chosen solvent The mixture to be separated is added on top of the slurry, solvent is then run through the column Different substances will interact with the stationary and mobile in different ways M ixture is separated depending on how strongly a component adsorbs to the stationary phase or how soluble they are in the mobile phase. Mobile phase – solvent (eluent) Stationary phase Sample Tap Component 2 – strongly adsorbed to stationary phase – takes longer to pass through the column Component 1 – Less strongly adsorbed to stationary phase, more soluble in solvent – passes through the column quickly

11. Gas Chromatography (GC) Gas chromatography is an analytical method used to determine volatile components and their amounts in a mixture Stationary phase – packed column Mobile phase – inert carrier gas (helium) The recorder records the time taken for a compound to go through the column – retention time The area under the peak on the chromatogram is relative to the amount of substance in the sample

12. 1. Volatile mixture is injected into the GC 2 . Mixture is vaporised due to the heat of the oven 3. Carrier gas pushes the sample through the column 4. The detector records the time taken for a substance to go through the column which is sent to the computer 5 . A graph is plotted showing the time each components reached the detector Each component will move at different rates through the column Some components will bond to the stationary phase more strongly – have a greater retention time Some components will not bond to the stationary phase strongly – shorter retention time

13. Gas chromatography Gas chromatography set up Sample injected here Column Inside the gas chromatograph Chromatogram produced

14. GC-MS GC – MS (Gas chromatography – mass spectroscopy) A GC can be linked to a mass spectrometer Unknown mixtures can be processed to determine their components Combines the benefits of both GS and MS Mixture is separated by the gas chromatography A mass spectrum is then produced for each component Mass spectrum can be compared to a database by a computer to determine the compound

15. Chromatography applications Forensics – analysing blood or fibre samples Drug testing – test a persons breathe to see if they have consumed any alcohol or drugs Pharmaceutical industry – test the purity Separating enantiomers