F324 Chromatography

Chromatography

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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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 componentMass 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

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.

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 valuesCompares how far a component spot has moved compared to the solvent front:

Rf = distance moved by componentdistance moved by solvent front

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Definition: ADSORBED = weakly bonded to a surface

F324 Chromatography

IdentificationThe 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.

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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 worksi) solid stationary phaseThe 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 phaseDifferences 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.

MethodMixture 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

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 GCGC 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.

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Definition: Retention time is the time between injection of a sample and the appearance of a given peak in the detector signal.

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-MSCombining 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

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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 analysisGC-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 analysisUsed for monitoring and quantifying organic pollutants in the environment, e.g. quality of waste water or drinking water, pesticides in foods

airport securityOne of the methods used for detecting traces of explosives in luggage or on humans

space probesGC-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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