Swami Sivananda State Secondary School CHROMATOGRAPHY 2009 David PHILIPPE Swami Sivananda State Secondary School

CHROMATOGRAPHY Chromatography Is a technique used to separate and identify the components of a mixture. Methods involve a stationary phase and a mobile phase and works by allowing the molecules present in the mixture to distribute themselves between the stationary and the mobile medium. There are several forms of chromatography

CHROMATOGRAPHY Chromatography Is a technique used to separate and identify the components of a mixture. Methods involve a stationary phase and a mobile phase and works by allowing the molecules present in the mixture to distribute themselves between the stationary and the mobile medium. There are several forms of chromatography TYPE STATIONARY PHASE MOBILE PHASE paper solid (filter paper) liquid thin layer (tlc) solid (silica) liquid column solid (silica) liquid high pressure liquid (hplc) solid (silica) liquid gas liquid (glc) solid or liquid gas

PAPER CHROMATOGRAPHY Stationary phase chromatography paper Mobile phase suitable solvent (water, ethanol, organic solvent) Separation As 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.

PAPER CHROMATOGRAPHY Stationary phase chromatography paper Mobile phase suitable solvent (water, ethanol, organic solvent) Separation As 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 PAPER CHROMATOGRAPHY Stationary phase chromatography paper Mobile phase suitable solvent (water, ethanol, organic solvent) Separation As 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

PAPER CHROMATOGRAPHY Rf 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. Rf = distance travelled by the component = X distance travelled by the solvent Y Y X

PAPER CHROMATOGRAPHY Rf 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. Rf = distance travelled by the component = X distance travelled by the solvent Y Comparison can be a problem if… a) components have similar Rf values b) the unknown substance is new and there is no previous chemical to compare it with Y X

What if the substances you are interested in are colourless? In some cases, it may be possible to make the spots visible by reacting them with something which produces a coloured product. A good example of this is in chromatograms produced from amino acid mixtures. Suppose you had a mixture of amino acids and wanted to find out which particular amino acids the mixture contained. A small drop of a solution of the mixture is placed on the base line of the paper, and similar small spots of the known amino acids are placed alongside it. The paper is then stood in a suitable solvent and left to develop as before. In the diagram, the mixture is M, and the known amino acids are labelled 1 to 5. The position of the solvent front is marked in pencil and the chromatogram is allowed to dry and is then sprayed with a solution of ninhydrin. Ninhydrin reacts with amino acids to give coloured compounds, mainly brown or purple.

Producing a paper chromatogram Suppose you have three blue pens and you want to find out which one was used to write a message. Briefly describe how you will proceed. Answer on the next two pages

Procedure: Samples of each ink are spotted on to a pencil line drawn on a sheet of chromatography paper. Some of the ink from the message is dissolved in the minimum possible amount of a suitable solvent, and that is also spotted onto the same line. In the diagram, the pens are labelled 1, 2 and 3, and the message ink as M. The paper is suspended in a container with a shallow layer of a suitable solvent or mixture of solvents in it. It is important that the solvent level is below the line with the spots on it The container is covered in order to make sure that the atmosphere in the beaker is saturated with solvent vapour. Saturating the atmosphere in the beaker with vapour stops the solvent from evaporating as it rises up the paper.

As the solvent slowly travels up the paper, the different components of the ink mixtures travel at different rates and the mixtures are separated into different coloured spots. It is fairly easy to see from the final chromatogram that the pen that wrote the message contained the same dyes as pen 2. You can also see that pen 1 contains a mixture of two different blue dyes - one of which might be the same as the single dye in pen 3.

A two way paper chromatography Two way paper chromatography gets around the problem of separating out substances which have very similar Rf values. a chromatogram is made starting from a single spot of mixture placed towards one end of the base line. It is stood in a solvent as before and left until the solvent front gets close to the top of the paper.

What you do now is to wait for the paper to dry out completely, and then rotate it through 90°, and develop the chromatogram again in a different solvent. It is very unlikely that the two confusing spots will have the same Rf values in the second solvent as well as the first, and so the spots will move by a different amount.

How does paper chromatography work? Paper is made of cellulose fibres, and cellulose is a polymer of the simple sugar, glucose. The key point about cellulose is that the polymer chains have -OH groups sticking out all around them. The cellulose fibres attract water vapour from the atmosphere as well as any water that was present when the paper was made Suppose you use a non-polar solvent such as hexane to develop your chromatogram. Non-polar molecules in the mixture that you are trying to separate will have little attraction for the water molecules attached to the cellulose, and so will spend most of their time dissolved in the moving solvent. Molecules like this will therefore travel a long way up the paper carried by the solvent. They will have relatively high Rf values. On the other hand, polar molecules will have a high attraction for the water molecules and much less for the non-polar solvent. They will therefore tend to dissolve in the thin layer of water around the cellulose fibres much more than in the moving solvent. Because they spend more time dissolved in the stationary phase and less time in the mobile phase, they aren't going to travel very fast up the paper. The tendency for a compound to divide its time between two immiscible solvents (solvents such as hexane and water which won't mix) is known as partition. Paper chromatography using a non-polar solvent is therefore a type of partition chromatography.

THIN LAYER CHROMATOGRAPHY Stationary phase silica mounted on a glass plate Mobile phase suitable organic solvent Separation similar technique to paper chromatography Limitations similar to paper chromatography

COLUMN CHROMATOGRAPHY Stationary phase silica Mobile phase suitable organic solvent Separation components interact with the stationary phase to different extents A B B C

COLUMN CHROMATOGRAPHY Stationary phase silica Mobile phase suitable organic solvent Separation components 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

HIGH PRESSURE 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 phase silica Mobile phase suitable solvent Separation similar to column chromatography

HIGH PRESSURE 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 phase silica Mobile phase suitable solvent Separation similar 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

HIGH PRESSURE 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 phase silica Mobile phase suitable solvent Separation similar 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

GAS LIQUID CHROMATOGRAPHY (GLC) Stationary phase liquid adsorbed on an inert solid support Mobile phase gas 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.

GAS LIQUID CHROMATOGRAPHY (GLC) Retention time The 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.

GAS LIQUID CHROMATOGRAPHY (GLC) Retention time The 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 point high boiling point = long retention time

GAS LIQUID CHROMATOGRAPHY (GLC) Retention time The 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 point high boiling point = long retention time solubility in the liquid phase greater solubility = long retention time

GAS LIQUID CHROMATOGRAPHY (GLC) Retention time The 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 point high boiling point = long retention time solubility in the liquid phase greater solubility = long retention time ANIMATION

In the animation below the red molecules are more soluble in the liquid (or less volatile) than are the green molecules.

Injection port Recorder Oven Detector Column Nitrogen cylinder

Chromatogram of petrol Suggest identities of some of the unlabelled peaks.

GAS LIQUID CHROMATOGRAPHY (GLC) Detection • there are several ways to detect components • most involve destruction of the sample • one method is an FID - flame ionisation detector The FID • as a component exits, it is burned in a hydrogen flame • ions are produced in the flame • a detector produces an electric current • greater the amount of a component = larger current • the current can be represented by a chromatogram • as the component is destroyed, GCMS doesn’t use FID

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.

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

GAS CHROMATOGRAPHY – MASS SPECTROMETRY (GCMS) Process When 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

GAS CHROMATOGRAPHY – MASS SPECTROMETRY (GCMS) Process When 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

GAS CHROMATOGRAPHY – MASS SPECTROMETRY (GCMS) Process When 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

GAS CHROMATOGRAPHY – MASS SPECTROMETRY (GCMS) Process When 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

For more information, consult the notes on ‘Mass Spectrometry’ A MASS SPECTROMETER ANALYSER DETECTOR ION SOURCE 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 For more information, consult the notes on ‘Mass Spectrometry’

CHROMATOGRAPHY The End