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CHROMATOGRAPHY Chromatography is used to separate and analyse small amounts of mixtures Methods involve a stationary phase and a mobile phase. There are.

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Presentation on theme: "CHROMATOGRAPHY Chromatography is used to separate and analyse small amounts of mixtures Methods involve a stationary phase and a mobile phase. There are."— Presentation transcript:

1 CHROMATOGRAPHY Chromatography is used to separate and analyse small amounts of mixtures Methods involve a stationary phase and a mobile phase. There are several forms of chromatography

2 CHROMATOGRAPHY Chromatography is used to separate and analyse small amounts of mixtures Methods involve a stationary phase and a mobile phase. There are several forms of chromatography TYPE STATIONARY PHASEMOBILE PHASE papersolid (filter paper) liquid thin layer (tlc) solid (silica) liquid column solid (silica) liquid high performance liquid (hplc) solid (silica) liquid gas liquid (glc) solid or liquid gas

3 PAPER CHROMATOGRAPHY Stationary phasechromatography paper Mobile phasesuitable solvent (water, ethanol, organic solvent) SeparationAs the solvent moves up the paper it dissolves the components and moves them up the paper. The more soluble a component is, the further it moves. Place small a spot of the mixture to be analysed (and any possible component for comparison purposes) on the paper. Dip the paper in the solvent.

4 PAPER CHROMATOGRAPHY Stationary phasechromatography paper Mobile phasesuitable solvent (water, ethanol, organic solvent) SeparationAs the solvent moves up the paper it dissolves the components and moves them up the paper. The more soluble a component is, the further it moves. Place small a spot of the mixture to be analysed (and any possible component for comparison purposes) on the paper. Dip the paper in the solvent. Allow the solvent to rise up the paper. Each component dissolves in the solvent. Those which are more soluble travel further up the paper.

5 PAPER CHROMATOGRAPHY Stationary phasechromatography paper Mobile phasesuitable solvent (water, ethanol, organic solvent) SeparationAs the solvent moves up the paper it dissolves the components and moves them up the paper. The more soluble a component is, the further it moves. Place small a spot of the mixture to be analysed (and any possible component for comparison purposes) on the paper. Dip the paper in the solvent. Allow the solvent to rise up the paper. Each component dissolves in the solvent. Those which are more soluble travel further up the paper. Finished chromatogram

6 PAPER CHROMATOGRAPHY R f value Under similar conditions, a component should always travel at the same speed. Its identity can be found by comparing the distance it moves relative to the solvent. R f = distance travelled by the component = Y distance travelled by the solvent X X Y

7 PAPER CHROMATOGRAPHY R f value Under similar conditions, a component should always travel at the same speed. Its identity can be found by comparing the distance it moves relative to the solvent. R f = distance travelled by the component = Y distance travelled by the solvent X Comparison can be a problem if… a) components have similar R f values b) the unknown substance is new and there is no previous chemical to compare it with X Y

8 THIN LAYER CHROMATOGRAPHY Stationary phasesilica mounted on a glass plate Mobile phasesuitable organic solvent Separationsimilar technique to paper chromatography Limitationssimilar to paper chromatography

9 COLUMN CHROMATOGRAPHY Stationary phasesilica Mobile phasesuitable organic solvent Separationcomponents interact with the stationary phase to different extents A B B C

10 COLUMN CHROMATOGRAPHY Stationary phasesilica Mobile phasesuitable organic solvent Separationcomponents interact with the stationary phase to different extents Method a chromatography column is filled with solvent and silica drops of the mixture are placed on top of the silica - A the tap is opened to allow the solvent to flow out additional solvent is added on top to replace that leaving components travel through at different rates and separate - B batches of solvent are collected at intervals - C the solvent in each batch is evaporated to obtain components A B B C

11 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) A better form of column chromatography. Instead of draining down through the stationary phase, the solvent is forced through under high pressure. Stationary phasesilica Mobile phasesuitable solvent Separationsimilar to column chromatography

12 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) A better form of column chromatography. Instead of draining down through the stationary phase, the solvent is forced through under high pressure. Stationary phasesilica Mobile phasesuitable solvent Separationsimilar to column chromatography Method a sample is injected solvent and sample are pushed through under pressure different compounds have different retention times output can be detected by compounds absorbing UV can be connected to a mass spectrometer

13 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) A better form of column chromatography. Instead of draining down through the stationary phase, the solvent is forced through under high pressure. Stationary phasesilica Mobile phasesuitable solvent Separationsimilar to column chromatography Method a sample is injected solvent and sample are pushed through under pressure different compounds have different retention times output can be detected by compounds absorbing UV can be connected to a mass spectrometer Advantages it is fast the path is short - usually under 30cm it gives better separation

14 GAS LIQUID CHROMATOGRAPHY (GLC) Stationary phaseliquid adsorbed on an inert solid support Mobile phasegas Method a very small amount of a sample is injected into the machine the injector is contained in an oven the sample boils and is carried along a thin column by an inert carrier gas column contains a liquid stationary phase, adsorbed onto an inert solid the time taken to travel through the tube will depend on how much time is spent moving with the gas rather than being attached to the liquid.

15 GAS LIQUID CHROMATOGRAPHY (GLC) Retention timeThe time taken for a compound to travel through the column to the detector. It is measured from the time the sample is injected to the time its peak shows maximum height.

16 GAS LIQUID CHROMATOGRAPHY (GLC) Retention timeThe time taken for a compound to travel through the column to the detector. It is measured from the time the sample is injected to the time its peak shows maximum height. For a particular compound, the retention time depends on... boiling pointhigh boiling point = long retention time

17 GAS LIQUID CHROMATOGRAPHY (GLC) Retention timeThe time taken for a compound to travel through the column to the detector. It is measured from the time the sample is injected to the time its peak shows maximum height. For a particular compound, the retention time depends on... boiling pointhigh boiling point = long retention time solubility in the liquid phasegreater solubility = long retention time

18 GAS LIQUID CHROMATOGRAPHY (GLC) Retention timeThe time taken for a compound to travel through the column to the detector. It is measured from the time the sample is injected to the time its peak shows maximum height. For a particular compound, the retention time depends on... boiling pointhigh boiling point = long retention time solubility in the liquid phasegreater solubility = long retention time ANIMATION

19 GAS LIQUID CHROMATOGRAPHY (GLC) Interpretation each compound in the mixture will produce a peak the areas under the peaks are proportional to the amount of a compound retention times are used to identify compounds – they are found out by putting known compounds through the system under similar conditions The area under a peak is proportional to the amount present. Because each compound responds differently, the machine is calibrated beforehand to show the actual mount. Each component has a different retention time.

20 GAS CHROMATOGRAPHY – MASS SPECTROMETRY (GCMS) ProcessWhen a peak is detected in gas chromatography, some of the component is sent to a mass spectrometer A mass spectrometer has three main parts...

21 GAS CHROMATOGRAPHY – MASS SPECTROMETRY (GCMS) ProcessWhen a peak is detected in gas chromatography, some of the component is sent to a mass spectrometer A mass spectrometer has three main parts... Ioniser- the sample is bombarded with electrons and ionised - a positive molecular ion is formed - the molecular ion can break up into smaller ions - positive ions are accelerated towards the analyser

22 GAS CHROMATOGRAPHY – MASS SPECTROMETRY (GCMS) ProcessWhen a peak is detected in gas chromatography, some of the component is sent to a mass spectrometer A mass spectrometer has three main parts... Ioniser- the sample is bombarded with electrons and ionised - a positive molecular ion is formed - the molecular ion can break up into smaller ions - positive ions are accelerated towards the analyser Analyser- positive ions separate according to mass/charge ratio - higher mass/charge ratio = smaller deflection

23 GAS CHROMATOGRAPHY – MASS SPECTROMETRY (GCMS) ProcessWhen a peak is detected in gas chromatography, some of the component is sent to a mass spectrometer A mass spectrometer has three main parts... Ioniser- the sample is bombarded with electrons and ionised - a positive molecular ion is formed - the molecular ion can break up into smaller ions - positive ions are accelerated towards the analyser Analyser- positive ions separate according to mass/charge ratio - higher mass/charge ratio = smaller deflection Detector- records the identity and abundance of each ion - compounds have a unique mass spectrum - the final peak (molecular ion) gives the molecular mass

24 GAS CHROMATOGRAPHY – MASS SPECTROMETRY (GCMS) ProcessWhen a peak is detected in gas chromatography, some of the component is sent to a mass spectrometer A mass spectrometer has three main parts... Ioniser- the sample is bombarded with electrons and ionised - a positive molecular ion is formed - the molecular ion can break up into smaller ions - positive ions are accelerated towards the analyser Analyser- positive ions separate according to mass/charge ratio - higher mass/charge ratio = smaller deflection Detector- records the identity and abundance of each ion - compounds have a unique mass spectrum - the final peak (molecular ion) gives the molecular mass

25 A MASS SPECTROMETER For more information, consult the notes on ‘Mass Spectrometry’ ION SOURCE ANALYSER DETECTOR IONISATION gaseous atoms are bombarded by electrons from an electron gun and are IONISED sufficient energy is given to form ions of 1+ charge ACCELERATION ions are charged so can be ACCELERATED by an electric field DEFLECTION charged particles will be DEFLECTED by a magnetic or electric field DETECTION by electric or photographic methods

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