Chromatography

Chromatography is good for separating and identifying thingsChromotography is used to separate stuff in a mixture - once it's separated out, you can often identify the components. There are quite a few different types of chromatography, but they all have the same basic set up: A mobile phase: Where the molecules can move. This is a liquid or a gas. A stationary phase: Where the molecules can't move. This must be a solid or a liquid on a solid support. And they all have the same basic principle: The mobile phase moves through or over the stationary phase. The distance each substance moves up the plate depends on its solubility in the mobile phase and it's retention by th stationary phase. Components that are more soluble in the mobile phase will travel further up the plate. It's these difference in solubility and retention by the stationary phase that separate out the different substances.

Thin-Layer chromatography is a simple way of separating mixtures In thin-layer chromatography (TLC), the stationary phase is a thin layer of silica (silicon dioxide) or aluminia (aluminium oxide) fixed to a glass or metal plate. Draw a line in pencil near the bottom of the TLC plate (the baseline) and put a very small drop of each mixture to be separated on the line. Allow the spots on the plate to dry Place the plate in a beaker with a small volume of solvent (this is the mobile phase). The solvent level must be below the baseline so that it doesn't dissolve the sample away. The solvent will start to move up the plate. As it moves, the solvent carries the substances in the mixture with it - some chemicals will be carries faster than others and so travel further up the plate. Leave the beaker until the solvent has moved almost to the top of the plate. Then remove the plate from the beaker. Before it evaporates, use a pencil to mark how far the solvent travelled up the plate (this line is called the solvent front). Place the plate in the fume cupboard and leave it to dry. The fume cupboard will prevent any toxic or flammable fumes from escaping into the room. The result is called a chromatogram. You can use the positions of the spots on the chromatogram to identify the chemicals. It's a good idea to wear gloves when handling the plate to prevent contamination by substances on your hands.

Colourless chemicals are revealed using UV light or iodineIf the chemicals in the mixture are coloured (such as dyes that make up an ink) then you'll see them as a set of coloured dots at different heights on the TLC plate. But if they're colourless chemicals, such as amino acids, in the mixture, you need to find a way of making them visible: Many TLC plates have a special fluorescent dye added to the silica or aluminia layer that glows when UV light shines on it. You can put the plate under a UV lamp and draw around the dark patches to show where the chemicals are. Expose the chromatogram to iodine vapour (leaving the plate in a sealed jar with a couple of iodine crystals works). Iodine vapour is a locatingagent - it sticks to the chemicals on the plate and they'll show up as brown/purple spots.

The position of the spots on a plate can help to identify substancesIf you just want to know how many chemicals are present in a mixture, all you have to do is count the number of spots that form on the plate. But if you want to find out what each chemical is, you can calculate something called an Rf value. The formula for this is:Rf = distance travelled by the spot / distance travelled by the solvent Rf values aren't dependant on how big the plate is or how far the solvent travels - they're properties of the chemicals in the mixture and so can be used to identify those chemicals. This means you can look your Rf value up in a table of standard Rf values to identify what the substance is. BUT, if the composition of the TLC plate, the solvent or the temperature changes even slightly, you'll get a different Rf value. It's hard to keep the conditions identical. So, if you suspect that a mixture contains chlorophyll for example, it's best to put a spot of chlorophyll on the baseline of the same plate as the mixture and run them both at the same time.

Column chromatography is used to separate out solutionsColumn chromatography is mostly used for purifying an organic product. It involves packing a glass column with a slurry of an absorbent material such as aluminium oxide, coated with water. This is the stationary phase. The mixture to be separated is added to the top of the column and allows to drain down into the slurry. A solvent is then run slowly and continually through the column. This solvent is the mobile phase. As the mixture is washed through the column, its components separate out according to how soluble they are in the mobile phase and how strongly they are absorbed onto the stationary phase (retention). Each different component will spend some time adsorbed onto the stationary phase and some time dissolved in the mobile phase. The more soluble each component is in the mobile phase, the quicker it'll ass through the column.

Gas chromatography is used to separate mixtures of volatile liquids If you've got a mixture of volatile liquids then gas chromatography (GC) is they way to separate them out so that you can identify them. The stationary phase is a solid or a solid coated by a viscous liquid, such as an oil, packed into a long tube. The tube is coiled to save space and built into an oven. The mobile phase is an unreactive carrier gas such as nitrogen. Each component takes a different amount of time from being injected into the tube to being recorded at the other end. this i the retention time. Each separate substance will have a unique retention time - so you can use the retention time to identify the components of the mixture (you have to run a known sample under the same conditions for comparison). The area under each peak tells you the relative amount of each component that's present in the mixture. GC can be used to find the level of alcohol in blood or urine - the results are accurate enough to be used as evidence in court. It's also used to find the proportions of various esters in oils used in paints - this lets picture restorers known exactly what paint was originally used.

Mass spectrometry can be combined with gas chromatographyMass spectrometry is a technique used to identify substances from the mass/charge ratio. It is very good at identifying unknown compounds, but would give confusing results from a mixture of substances. Gas chromatography is very good at separating a mixture into its individual components, but not so good at identifying those components. If you put the two techniques together, you get an extremely useful analytical tool Gas chromatography-mass spectrometry (GC-MS) combines the benefits of gas chromatography and mass spectrometry. The sample is separated using GC, but instead of going to a detector, the separated components are fed into a mass spectrometer. The spectrometer produces a mass spectrum for each component, which can be used to identify each one and show what the original sample consisted of. The advantage of this method over normal GC is that the components separated out by the chromatography can be positively identified, which can be impossible from a chromatogram alone.Computers can be used to match up the mass spectrum for each component of the mixture against a database, so the whole process can be automated.