Chromatography 4.3.1
Chromatography is used to separate mixtures of substances into their
components.
Student Activity
Choose a separation technique for the getting the bold component out of the
mixture. These might be useful but you could think of other techniques too.
evaporation, decanting, chromatography, distillation, chromatography,
filtration.
Mixture
Sand and water
NaCl and water
Ethanol and hexane
KOH and water
Hexane and water
C6H12O6 and NaCl
Separation Technique
There are two types of chromatography that we will look at:
1) Thin Layer Chromatography (TLC) and 2) Gas Chromatography (GC or
GLC).
They both have a stationary phase: a solid (TLC), or a liquid/solid supported
on a solid (GLC) and a mobile phase: a liquid or a gas. The mobile phase
flows through the stationary phase and carries the components of the mixture
with it. Different components travel at different rates.
One type of GC (called GLC) separates by partition/relative solubility
whereas TLC and another type of GC separate by adsorption.
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Remember paper chromatography from school or A level Biology?
The chromatogram was run using water as the solvent. Use the
chromatogram to answer the following questions:
1. How many compounds are in the purple ink?
2. Which coloured compound(s) are contained in the felt-tip pen? How do
you know this?
Measuring Rf values
Measurements are often taken from the chromatogram in order to help
identify the compounds present. These measurements are the distance
travelled by the solvent, and the distance travelled by individual spots.
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The Rf value for each dye can be worked out using the formula:
The Rf value for a particular component will always be the same if you do it
under the same conditions. However, if temperature or the solvent changes,
the Rf value will be different.
3. Estimate the Rf values of the three spots in the felt-tip pen. Show your
working (you may do this on the diagram). Use a ruler!
4. Ethanol is sometimes used as the solvent when running chromatograms
on inks. Explain why you may need to use ethanol instead of water.
NB You will not be asked about paper chromatography in your exams,
1. Thin Layer Chromatography:
You will watch some material to explore how this works and how to do this.
http://www.chem-ilp.net/labTechniques/TLC.htm
When you think you have seen and heard enough, answer the following
questions.
a) What do we mean by the stationary phase?
b) What is the stationary phase in TLC?
c) Why are the solvents (moving phase) usually organic?
d) Why is UV light used?
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e) Give an example of a ‘chemical dip’, your teacher will mention others
How it works
As the solvent begins to travel up the plate (by capillarity) it dissolves the
compounds in the spot. The compounds present will then get carried up the
chromatography plate with the solvent.
How fast the compounds get carried up the plate depends on two things:


How soluble the compound is in the solvent. This depends on how
much attraction there is between the molecules of the compound and
those of the solvent. The more attraction there is the more soluble the
compound.
How much the compound adsorbs onto the stationary phase. This
depends on how much attraction there is between the molecules of the
compound and the stationary phase.
The stationary phase - silica gel
Silica gel is a form of silicon dioxide (silica). The silicon atoms are joined via
oxygen atoms in a giant covalent structure. However, at the surface of the
silica gel, the silicon atoms are attached to -OH groups.
At the surface of the silica gel you have Si-O-H bonds instead of Si-O-Si
bonds. The diagram shows a small part of the silica surface.
The surface of the silica gel is very polar because of the -OH groups so it can
form hydrogen bonds with suitable compounds around.
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Student Activity: complete the table to show the intermolecular forces
between molecules of these individual substances. (You can choose from van
der Waal’s, permanent dipole or hydrogen bonding)
Compound
ethanol
hexane
2,3-dimethylbutane
butanoic acid
propanone
cyclohexane
2- chloropropane
propan-2-ol
methylamine (CH3NH2)
pentanoic acid
but-2-ene
Type of interaction
Interactions with the stationary phase:
Suppose the original spot contained two compounds - one of which can form
hydrogen bonds, and one of which can only take part in weaker van der
Waal’s interactions.
The one which can hydrogen bond will adsorb to the surface of the silica gel
more firmly than the other one. (Adsorption is the name given to one
substance forming some sort of bonds to the surface of another one and it is
not permanent).
Obviously the compound can only travel up the plate during the time that it is
dissolved in the solvent. While it is adsorbed on the silica gel, it is temporarily
stopped - the solvent is moving on without it. The more strongly a compound
is adsorbed, the less distance it can travel up the plate. The compound which
can form hydrogen bonds will adsorb more strongly than the one dependent
on van der Waal’s interactions.
A solid stationary phase separates components by adsorption.
Student Activity: Underline words in the above section meaning:
A substance forming bonds to the surface of another substance. An
example of a stationary phase. Interactions formed by hydrocarbon
compounds. The mobile phase.
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2. Gas Chromatography
http://www.youtube.com/watch?v=08YWhLTjlfo
One kind of gas chromatography uses an inert solid support and an inert gas;
this is adsorption chromatography just like TLC but using a gas as the solvent
instead of a liquid.
In another kind of gas chromatography, the mobile phase is a gas such as
helium and the stationary phase is a high boiling point liquid adsorbed onto an
inert solid (GLC).
How fast a particular compound travels through the machine will depend on
how much of its time is spent moving with the gas, as opposed to being
‘attached’ to the liquid.
This kind of GC separates out compounds based on a physical process called
partition (or relative solubility) - a measure of how soluble the substance is in
the stationary phase compared to the mobile phase.
Your teacher will show you a demonstration to explain an example of this idea
of relative solubility using chemistry from Unit 1 at AS.
cyclohexane/water and iodine, iodine dissolves in both solvents – yellow/red
in water layer and purple/pink in the cyclohexane layer (top)
Retention time
The time taken for a compound to from travel through the column to the
detector from the point of injection is known as its retention time.
Different compounds have different retention times. For a particular
compound, the retention time will vary with its solubility in the liquid phase
and the temperature of the column.
Compounds which spend most of their time in the gas phase will pass quickly
through the column. The more soluble a compound is in the mobile phase the
shorter the retention time. The more soluble a compound is in the stationary
phase, the longer it will take and therefore the longer the retention time.
Here is a practical application of the use of GC
http://www.edusolns.com/gc/gctutorial/
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Interpreting the results:
Two things are considered when looking at a gas chromatograph.
1) Retention times - used to help identify the compounds present by
comparing them with retention times for pure, known samples.
2) The area under the peaks - used to determine the relative amount of
each compound in the sample (area under the peak is proportional to
the quantity).
This GC shows the analysis of
compounds dissolved in hexane from a
cannabis leaf. Note that the retention
time of 0 s is on the right and that the
chromatograph is read from right to left.
X
Compounds with the longest retention
time will appear on the left of the
chromatograph. These are the
compounds which are more soluble in
the stationary (liquid) phase.
Y
time/s
Limitations
1. Problems arise when using chromatography to analyse similar compounds
as they will often have similar retention times or Rf values.
2. Reference retention times are impossible when dealing with unknown
substances.
3. Retention times are dependent on temperature, pressure, choice of gas
and individual machine.
Combining Mass Spectroscopy and Chromatography
Analytical techniques in chemistry are much more powerful when used
together. Chromatography can be used to separate compoudsand mass
spectroscopy will identify them from the molecular ion (M+) peak.
This technique of combining mass spectroscopy with gas chromatography
(GC-MS) is often used in forensics, environmental analysis, airport security
and even on space probes.
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Questions
1. Identify the stationary and mobile phases used in GC and TLC.
GC
TLC
Mobile Phase
Stationary Phase
2. Predict how raising the temperature might affect the retention time of a
particular component in gas chromatography. Two things!
3. Two esters, CH3(CH2)2COO(CH2)3CH3 and CH3(CH2)2COOCH2CH3,
contribute to the odour of pineapple. A food scientist analysed a sample of
pineapple essence by separating the two esters using gas chromatography,
GC, and measuring their retention times.
(a) (i) State what is meant by retention time.
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(ii) Explain the possible limitations of GC in separating the two esters.
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4. Amino acids are found in human sweat. A student had read that
chromatography could be used to separate and identify the amino acids
present in human sweat. The student used Thin-Layer Chromatography (TLC)
to separate the amino acids in a sample of human sweat and discovered that
three different amino acids were present.
(i) Name the process by which TLC separates amino acids.
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(ii) The chromatogram was treated to show the positions of the separated
amino acids. Explain how the student could analyse the chromatogram to
identify the three amino acids that were present.
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(iii) Several amino acids have structures that are very similar.
Suggest why this could cause problems when using TLC to analyse mixtures of
amino acids.
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5. Here is a print out from a GC machine:-
a) which component is held held least strongly in the column? Explain.
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b) Both caffeine and theobromine are present in the sample in the same
proprtions. Explain how this is shown on the graph.
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c) Suggest why Gas Chromatography is not generally suitable for
analysing a mixture of dyes.
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