Transcript CHROMAPP - davidphilippe

CHROMATOGRAPHY
2009
Swami Sivananda State Secondary School
David PHILIPPE
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
paper
solid (filter paper)
thin layer (tlc)
MOBILE PHASE
liquid
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.
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.
Y
Rf =
distance travelled by the component =
distance travelled by the solvent
X
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.
Y
Rf =
distance travelled by the component =
distance travelled by the solvent
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
X
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
key point about cellulose is that the polymer chains have -OH
sugar, glucose. The
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
A
B
Separation
components interact with the stationary
phase to different extents
B
C
COLUMN CHROMATOGRAPHY
Stationary phase
silica
Mobile phase
suitable organic solvent
A
B
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
B
•
•
•
•
C
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
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
solubility in the liquid phase
high boiling point = long retention time
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
solubility in the liquid phase
high boiling point = long retention time
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.
Each component has a different retention time.
Because each compound
responds differently, the
machine is calibrated
beforehand to show the
actual mount.
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
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