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F324 Chromatography

Types of chromatography describe chromatography as an analytical technique that separates components in a mixture between a mobile phase and a stationary phase. Paper chromatography can be used to illustrate the separation process. state that: (i) the mobile phase may be a liquid or a gas (ii) the stationary phase may be a solid (as in thin-layer chromatography, TLC) or either a liquid or solid on a solid support (as in gas chromatography, GC) (iii) a solid stationary phase separates by adsorption (iv) a liquid stationary phase separates by relative solubility explain the term Rf value, and interpret one-way chromatograms in terms of Rf values explain the term retention time, and interpret gas chromatograms in terms of retention times and the approximate proportions of the components of a mixture explain that analysis by gas chromatography has limitations, eg: (i) similar compounds often have similar retention times (ii) unknown compounds have no reference retention times for comparison Combining mass spectrometry with chromatography explain that mass spectrometry can be combined with chromatography: (i) to provide a far more powerful analytical tool than from chromatography alone (ii) to generate mass spectra which can be analysed or compared with a spectral database by computer for positive identification of a component Mass spectrometry is used with GC as GC-MS and with high pressure liquid chromatography as HPLC-MS; separated components are directed into the mass spectrometer. You may be expected to interpret provided gas chromatograms and mass spectra state the use of GC-MS in analysis, eg in forensics (including use of evidence in courts), environmental analysis, airport security and space probes

Chromatography separates the components in a mixture. We do this: - to find out how many components there are - to match the components with known reference materials - so that we can use other analytical techniques to individually identify each component Advantages: separates out components with similar physical properties in complex mixtures also versatile and not as expensive as NMR Disadvantage: Not very good at identifying anything unambiguously In any type of chromatography, separation occurs because there are two phases. A mobile phase, which sweeps the mixture in a definite direction over a stationary phase which is fixed in position, and which slows down each compound in the mixture by a different amount, separating them.

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F324 Chromatography

Thin Layer Chromatography (TLC)

The stationary phase is a solid, a thin layer of e.g. alumina (Al2O3) or silica gel (SiO2) coated on a flat inert support usually a sheet of glass or plastic. The mobile phase is a liquid solvent which moves vertically up the TLC plate. The components in the mixture are dissolved into the mobile phase and carried over the stationary phase. They are slowed down in their movement up the plate because they become adsorbed on the surface of the stationary phase then desorbed again continually. The more strongly it is adsorbed, the more time it will spend on the stationary phase, and the more slowly it will move. The more soluble a substance is in the mobile phase, the more easily it will be desorbed and are swept along more quickly.

Definition: ADSORBED = weakly bonded to a surface Used for: Low volatility compounds, such as solids, which can be dissolved in a solvent the solvent will need to be similar to the substances to be identified so they will dissolve. e.g. amino acids in a protein which has been broken down; pigments in a paint; forensics - identification of cannabis Method a spot of the dissolved sample mixture is dotted on the pencil baseline the spot allowed to dry, then the TLC plate is placed in a jar with a little solvent the solvent rises, meets the sample, the components of the mixture are swept up the plate and separated the solvent is allowed to rise near the top of the plate, then the position of the solvent front is marked the positions of the spots are marked if colourless, a locating agent (such as ninhydrin) or UV light may be used to show up each spot the chromatogram is interpreted using the Rf value for each spot Rf values Compares how far a component spot has moved compared to the solvent front: Rf = distance moved by component distance moved by solvent front

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F324 Chromatography Identification The Rf value can be used to identify a component by comparing with a database of known Rf values of pure substance (measured under the same conditions with the same solvent). A chromatogram of the unknown mixture is often run alongside samples of known pure substances (references) thought to be in the mixture. Limitations chemically similar compounds may have similar Rf values unknown compounds have no Rf values known for comparison may be difficult to find a solvent which separates all the components in the mixture trial and error, and perhaps a mixture of solvents, will be required. may tell you how many components are present, and Rf values may suggest what they may be, but DOES NOT give information about how much of each component is present.

Gas Chromatography (GC)

Very commonly-used instrumental technique, often fully automated. Used for: Separating volatile components in a mixture (gases, liquids, occasionally volatile solids) so particularly good for many organic compounds with low boiling points and that evaporate easily. e.g. components in petrol/fuels; alcohols and other organics present in beer, whiskey etc. Can also be used medically for dissolved gases in blood. Basic components carrier gas supply injection port chromatography column detector and computer/data system/recorder

The chromatography column is a capillary tube. It is very long (up to 30m) and coiled up to fit inside a temperature-controlled oven which is needed to control conditions. In GC the stationary phase may be liquid coated on the surface of an inert solid support coating the inner walls of the capillary tube. The liquid needs to be inert and non-volatile (high boiling point), so a long chain alkane is often used. The stationary phase could also be a solid supported on the inner walls of the capilliary tube. Suitable solids include silicone polymers. Page 3

F324 Chromatography In GC, different stationary phase substances are chosen for separating different types of compounds. The mobile phase is a carrier gas which must be inert, and which flows through the column carrying the sample mixture over the stationary phase. He or N2 is usually used. How it works i) solid stationary phase The vapourised components repeatedly adsorb onto the surface of the solid stationary phase, staying bound for a time which depends on the strength of their interaction with this phase. The more polar they are, the more strongly they adsorb and the slower they move through the capilliary. ii) liquid stationary phase Differences in solubility in the liquid forming the stationary phase, and volatility cause separation. The more soluble each component is in the liquid stationary phase, the more time it will spend dissolved and the more slowly it will travel through the capilliary. The more volatile the component is, the more rapidly it will leave solution and be swept along by the carrier gas. Method Mixture injected into the GC where it is vaporised and the mobile carrier gas carries it into column time of injection recorded Components carried through column (at different rates depending on solubility/adsorption) Separation can be improved by using different oven temperatures and different flow rates for the carrier gas. Each component leaves the column and passes though the detector at a different time, which is recorded. Identification of components is done on the basis of retention time: Retention time Definition: Retention time is the time between injection of a sample and the appearance of a given peak in the detector signal. Each component has a characteristic retention time (for given stationary phase, temperature, gas flow rate etc.). Retention times can be compared to a database of known values for pure reference compounds to aid identification of unknowns. Quantitative GC GC also tells us the relative amounts of each component in the mixture. The area under each peak is proportional to the amount of that compound in the sample. Page 4

F324 Chromatography e.g.

There are several methods of estimating the area under a peak, but a simple approximation is to measure the height of the peak and to multiply this by the width of the peak at half the height: area = height x width at half height In the example above we can see that the major component is pentan-2-one and the component present in the smallest proportion is ethanal. By estimating the peak areas we could also say how much more pentan-2-one there is than ethanal. Limitations of GC potentially thousands of substances may have same retention time, peak shape etc. so GC does not positively identify most components. It is more useful when (most of) the components and their retention times are known not all substances in the sample will necessarily be separated and detected small peaks can be hidden under larger ones unknowns have no reference retention time Advantages over TLC more rapid quantitative smaller quantities of materials may be detected GC results are often used in law courts, and any doubt about the method or reliability of the results will cast doubt on the evidence so GC is used in combination with other techniques. GC-MS Combining the two techniques uses the strengths of both. The output from the GC column is fed into a Mass Spectrometer, which acts as the detector. - GC can separate the components in a mixture and quantify the relative amounts of each present, but not identify them conclusively Page 5

F324 Chromatography - MS can provides a characteristic fragmentation pattern for each component which provides detailed structural information and which allows the component to be identified by computer using a spectral database. MS can't measure how much of each component in the mixture. Applications: forensics and drug analysis GC-MS can be used to analyse minute samples from scene of crime e.g. hair, skin flakes, dried blood flakes etc. The MS can identify specific substances such as illegal drugs in these samples, and can do so many days after the drugs have been taken. environmental analysis Used for monitoring and quantifying organic pollutants in the environment, e.g. quality of waste water or drinking water, pesticides in foods airport security One of the methods used for detecting traces of explosives in luggage or on humans space probes GC-MS can be ultra-miniaturised suitable for remote/automatic operation on space probes. Mars missions used GC-MS for analysing planetary material, and it was used for sampling Venus's atmosphere. Huygens probe landed a GC-MS on Saturn's moon, Titan, which provided direct evidence for methane on this moon's surface.

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