ChromatographyChromatography

Chapter 6Chapter 6

ChromatographyChromatography

A technique used to separate the substances present in A technique used to separate the substances present in a mixture.a mixture.Chromatography can be used for both qualitative and Chromatography can be used for both qualitative and quantitative analysisquantitative analysisIt can also be used to determine the identity of a It can also be used to determine the identity of a substance.substance.Chromatography applications include the identification Chromatography applications include the identification of:of:– Drugs present in bloodDrugs present in blood– Sugars in fruit juiceSugars in fruit juice– Hydrocarbons in oilHydrocarbons in oil– Pollutant gases in exhaust fumesPollutant gases in exhaust fumes

How does chromatography work?How does chromatography work?

There are many different methods of There are many different methods of chromatography, but all methods have:chromatography, but all methods have:– A A stationarystationary phase phase– A A mobilemobile phase (or moving phase) phase (or moving phase)

Turn to page 62 and look at figure 6.1Turn to page 62 and look at figure 6.1

What do you think is the stationary phase?What do you think is the stationary phase?

Different components of a mixture will be Different components of a mixture will be attracted (adsorb to) the stationary and mobile attracted (adsorb to) the stationary and mobile phases to different extents.phases to different extents.

Chromatography uses these differences in Chromatography uses these differences in attraction to separate the components.attraction to separate the components.

The moving fluid, referred to as the mobile The moving fluid, referred to as the mobile phase, passes over the stationary phase. phase, passes over the stationary phase.

The components of the mixture are separated The components of the mixture are separated according to their relative attractions to the according to their relative attractions to the mobile and stationary phases.mobile and stationary phases.

How does chromatography work?How does chromatography work?

The rate of movement of each component The rate of movement of each component depends mainly upon depends mainly upon – How strongly it adsorbs onto (is attracted to) How strongly it adsorbs onto (is attracted to)

the stationary phase.the stationary phase.– How readily it dissolves in the mobile phaseHow readily it dissolves in the mobile phase

How does chromatography work?How does chromatography work?

Paper and Thin-layer Paper and Thin-layer chromatography (TLC)chromatography (TLC)

TLC is similar to paper chromatography which TLC is similar to paper chromatography which you have done in junior scienceyou have done in junior science

Paper chromatography uses high quality Paper chromatography uses high quality absorbent paper (filter paper) as the stationary absorbent paper (filter paper) as the stationary phasephase

TLC uses a thin layer of fine powder such as TLC uses a thin layer of fine powder such as alumina, spread on a glass or plastic plate alumina, spread on a glass or plastic plate

Both paper and TLC are good for qualitative Both paper and TLC are good for qualitative analysis only.analysis only.

TLC - ProcedureTLC - Procedure

A solution of the sample to be analysed is made A solution of the sample to be analysed is made up up As small a spot as possible is place onto the end As small a spot as possible is place onto the end of the chromatography plateof the chromatography plate– This spot is called the originThis spot is called the origin

The plate is then placed into a container with the The plate is then placed into a container with the edge of the plate submerged in solvent (the edge of the plate submerged in solvent (the origin should be above the solvent)origin should be above the solvent)As the solvent rises up the plate the components As the solvent rises up the plate the components of each sample separateof each sample separate

TLCTLC

The different The different components of a components of a mixture will move at mixture will move at different rates different rates depending on relative depending on relative strength of attractions strength of attractions to the stationary and to the stationary and mobile phases.mobile phases.What is more strongly What is more strongly attracted to the attracted to the stationary phase?stationary phase?

Interpreting chromatograms of TLCInterpreting chromatograms of TLC

A chromatogram is the pattern of bands or A chromatogram is the pattern of bands or spots formed on the plate in TLCspots formed on the plate in TLCThe identity of the chemicals in the mixture The identity of the chemicals in the mixture can be identified in two wayscan be identified in two ways– 1. running standards of known chemicals on 1. running standards of known chemicals on

the same chromatogram as the unknown the same chromatogram as the unknown samplesample

– 2. calculating R2. calculating Rff values of the samples values of the samples

Which method would be more effective??Which method would be more effective??

RRff values values

RRff values will always be less than one values will always be less than one

The component most strongly adsorbed The component most strongly adsorbed onto the stationary phase moves the onto the stationary phase moves the shortest distance and has the lowest Rshortest distance and has the lowest Rff valuevalue

By comparing the RBy comparing the Rff values of values of components of a mixture with the Rcomponents of a mixture with the Rff values of known substances under values of known substances under identical conditions, the compounds identical conditions, the compounds present in a mixture can be identifiedpresent in a mixture can be identifiedWhat issues can occur with this methodWhat issues can occur with this method

Rf =Distance moved from origin by componentDistance moved from origin by solvent

Your TurnYour Turn

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Question 1Question 1

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Question 5bQuestion 5b

Column ChromatographyColumn ChromatographyThe stationary phase is a solid, or a solid The stationary phase is a solid, or a solid that has been thinly coated in a viscous that has been thinly coated in a viscous liquid and packed into a glass column.liquid and packed into a glass column.The sample is applied carefully to the top The sample is applied carefully to the top of the packing and a solvent, which acts as of the packing and a solvent, which acts as the mobile phase, is dripped slowly on to the mobile phase, is dripped slowly on to the column from a reservoir above.the column from a reservoir above.A tap at the bottom of the column allows A tap at the bottom of the column allows the solvent, which is called the eluent, to the solvent, which is called the eluent, to leave the column at the same rate as it leave the column at the same rate as it enters it at the other end.enters it at the other end.There are two techniques based on There are two techniques based on column chromatography, high performance column chromatography, high performance liquid chromatography (HPLC) and gas liquid chromatography (HPLC) and gas chromatography.chromatography.

HPLC and GCHPLC and GC

HPLC and GC chromatograms represent the HPLC and GC chromatograms represent the qualitative and quantitative aspects of qualitative and quantitative aspects of chromatography.chromatography.

– Number of peaks – mixture separated into three Number of peaks – mixture separated into three different compoundsdifferent compounds

– Retention time – identifies each component – Retention time – identifies each component – qualitative analysisqualitative analysis

– Area under each peak – relative amount of each Area under each peak – relative amount of each compound – quantitative analysiscompound – quantitative analysis

Retention time (Rt)

High performance liquid High performance liquid chromatography (HPLC)chromatography (HPLC)

This method is used routinely for This method is used routinely for pharmaceutical and industrial analysis. pharmaceutical and industrial analysis.

It allows extremely sensitive analysis of a It allows extremely sensitive analysis of a wide range of compounds.wide range of compounds.

It can be used to detect barbiturates in the It can be used to detect barbiturates in the blood.blood.

HPLCHPLC

There are many ways in which HPLC differs There are many ways in which HPLC differs from tradition chromatography:from tradition chromatography:– The size of the particles in the solid stationary phase The size of the particles in the solid stationary phase

is often 10-20 times smalleris often 10-20 times smaller– The very small size of these particles allows for more The very small size of these particles allows for more

frequent adsorption and desorption of the frequent adsorption and desorption of the components, giving better separationcomponents, giving better separation

– The small particles size creates considerable The small particles size creates considerable resistance to the flow of the mobile phase so the resistance to the flow of the mobile phase so the solvent is pumped through under high pressuresolvent is pumped through under high pressure

– A range of solids are available for use in HPLC A range of solids are available for use in HPLC columns, some with chemicals specially bonded to columns, some with chemicals specially bonded to their surfaces to improve the separation of particular their surfaces to improve the separation of particular classes of compoundsclasses of compounds

HPLCHPLC

The detector is usually a UV lightThe detector is usually a UV light– Many organic compounds absorb UV light Many organic compounds absorb UV light – The amount of light received by the The amount of light received by the

detector is recorded on a chart that detector is recorded on a chart that moves at constant speedmoves at constant speed

– This resulting trace is the chromatogramThis resulting trace is the chromatogramThe time taken for a component to pass The time taken for a component to pass through the column is called the retention through the column is called the retention time, Rtime, Rtt

– The RThe Rtt are used to identify the are used to identify the components associated with the peaks on components associated with the peaks on a chromatogram.a chromatogram.

The relative amounts of each component The relative amounts of each component in a mixture may be determined by in a mixture may be determined by comparing the areas under each peak comparing the areas under each peak with areas under peaks for standard with areas under peaks for standard samplessamples

Gas Chromatography (GC)Gas Chromatography (GC)

This is the most sensitive chromatographic This is the most sensitive chromatographic techniquetechnique

It is capable of detecting as little as 10It is capable of detecting as little as 10-12-12g g of a compoundof a compound

It is limited to compounds that can be It is limited to compounds that can be easily vaporised without decomposing.easily vaporised without decomposing.

Urine samples taken from athletes for drug Urine samples taken from athletes for drug tests are analysised by GCtests are analysised by GC

GCGC

Gas chromatography has the following featuresGas chromatography has the following features– The mobile phase is a gas, usually nitrogen, called The mobile phase is a gas, usually nitrogen, called

the carrier gasthe carrier gas– A small amount of sample is injected into the top of A small amount of sample is injected into the top of

the column through an injection portthe column through an injection port– The injection port is heated to a temperature sufficient The injection port is heated to a temperature sufficient

to instantly vaporise the sample, which is then swept to instantly vaporise the sample, which is then swept into the column by the carrier gas.into the column by the carrier gas.

– The column is in a loop, this is because of the fast The column is in a loop, this is because of the fast moving gaseous phase. So the column must be moving gaseous phase. So the column must be longer than in HPLC to allow for effective interaction longer than in HPLC to allow for effective interaction with the stationary phasewith the stationary phase

– The column is mounted in an oven and heatedThe column is mounted in an oven and heated

Interpreting ChromatogramsInterpreting Chromatograms

The time a component takes to pass through the The time a component takes to pass through the column is called the retention time, Rt.column is called the retention time, Rt.The same compound will give the same The same compound will give the same retention time if the conditions (temp, mobile retention time if the conditions (temp, mobile phase, stationary phase, flow rate, pressure etc) phase, stationary phase, flow rate, pressure etc) remain the same. remain the same. Each component forms one peak, however it is Each component forms one peak, however it is possible for a number of peaks to coincide and possible for a number of peaks to coincide and be indistinguishable.be indistinguishable.Look at page 69.Look at page 69.

Figure 6.12 Chromatogram of a reference sample containing a mixtureof butane, 2-methylbutane, hexane, benzene and 2-methylhexane.

Figure 6.11 Gas chromatogram of a petrol sample.

Figure 6.13 Petrol sample spiked with benzene.

Worked Example 6.3Worked Example 6.3Page 69Page 69

What is the concentration of the sample of What is the concentration of the sample of benzene?benzene?

Your Turn: Try question 14 on page 74

Chapter homeworkChapter homework

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Question 7 and 9Question 7 and 9

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Question 17Question 17

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Question 20 and 21Question 20 and 21