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BTEC APPLIED SCIENCE UNIT 2 - Learning aim C
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C: Undertake chromatographic techniques to identify components in mixtures. Grade Distinction.
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Emilia HawkinsUnit 2: Practical Scientific Procedures and Techniques
C: Undertake chromatographic techniques to identify components in mixtures
Explanation of chromatographic techniques and their suitability
Paper chromatography is used to separate dried liquid samples (e.g., ink, lipstick and fabric dyes)
which are dissolved in a solvent and then allowed to travel up paper using a solvent to identify their
chemical make-up. It can be done using a polar solvent (e.g. water) or non-polar solvent (e.g.
hexane). The chemical to be tested is dotted onto the bottom of a piece of chromatography paper.
The chromatography paper (stationary phase) is then placed into a liquid solvent (mobile phase). To
determine the chemicals in tested samples, comparisons can be made between the results of known
chemicals and the results of the unknown samples. As the solvent migrates up the chromatography
paper, dots will appear and the further a dot is from starting line the higher the R f value is. When
Chromatography is done with amino acids, the dots are not visible, but by spraying the paper with
ninhydrin or putting the paper under ultraviolet light you can see where the spots are. Ninhydrin
can be used as it reacts with the amino acids when it is sprayed onto the dried chromatograph, this
produces purple or brown coloured compounds on the paper. The final result, when using paper
chromatography is piece of chromatography paper with a number of dots that show the separated
compounds in a mixture.
o Advantages and disadvantages of paper chromatography:
Advantages Disadvantages
Can quickly separate a mixture. Less accurate compared to other forms of
chromatography.
Simple and easy to set up. Cannot separate a complex mixture.
Cost-effective. Results cannot be kept for long time periods.
Thin layer chromatography (TLC) is used to separate mixtures by coating a sheet of plastic or glass
with a layer of absorbent material. The stationary phase is usually silica gel or alumina, that is mixed
with a substance that glows under ultraviolet light, and the mobile phase is a liquid solvent. The
chemical is dotted onto the bottom of the sheet and then placed into a liquid solvent. The mobile
phase moves through the stationary phase and carries the mixture with it. If the spots produced on
the final chromatogram are predicted to be colourless, the stationary phase is mixed with a
substance that glows under ultraviolet light. This allows you to shine UV light onto the plate so the
spots will appear as dark patches, allowing you to visibly mark where they are to calculate R f values.
Alternatively, you could make the spots visible by reacting them with a chemical that has a coloured
product. Ninhydrin can be reacted with amino acids and sprayed onto the plate to produce coloured
spots which are usually brown or purple. The final result of thin layer chromatography is a plate
showing spots that have been separated from a mixture.
o Advantages and disadvantages of thin layer chromatography:
Advantages Disadvantages
Simple and easy to set up. Results are difficult to reproduce.
Can separate complex mixtures. Only qualitive analysis is possible.
Only requires a small sample size. Results can be affected by external factors such
as temperature and humidity.
, Emilia Hawkins
Column chromatography is used for the purification of biomolecules where they are separated using
their level of affinity to the mobile and stationary phase. This method of chromatography is done on
a much larger scale in comparison to paper or thin layer chromatography. Column chromatography
uses a vertical glass column that has been filled with silica gel or alumina, which has been saturated
with a solvent, and has a layer of glass wool at the bottom. The solution can be added to the top of
the column, and it will flow down the column to allow the mixture to move downwards. More
solvent is added into the top of the column to ensure that the silica or alumina doesn’t dry out as
the mixture is separating. Different chemicals flow through the column at different rates and
therefore reach the bottom at different times. There is also a tap at the bottom of the column to
allow the sample to pass through once it has been separated. When the stationary phase is polar
and the mobile phase is non-polar, the chemical in the sample that is less polar will take more time
to travel down the column, as it isn’t reacting as quickly with the mobile phase. Chemicals that are
more polar will pass through the column quicker and have a shorter retention time. The result of
column chromatography is liquid compounds that have been separated and collected into beakers.
o Advantages and disadvantages of columnar chromatography:
Advantages Disadvantages
Can separate complex mixtures. A time-consuming process.
The separated sample can be reused. A complicated process when automated.
No limit to the volume of mixture that can be It’s expensive as it uses large amounts of
separated. solvent.
Gas chromatography can be used to separate individual components in a mixture and the determine
how much of each chemical is present. The stationary phase is liquid that has a high boiling point
which has been absorbed onto a solid. The mobile phase in most other forms of chromatography is a
liquid but for gas chromatography it is an inert or noble gas. The time taken for the compounds to
travel through the column depends on how much time it spends moving with the gas rather than
being attached to the liquid. The column used in gas chromatography is made of stainless steel and
is 1-4 metres long, with a diameter of 4mm. This column is filled with a diatomaceous earth and is
coated with a high boiling liquid (stationary phase). It is heated to between 50 oC and 250oC but is
always kept cooler than the injector oven where the mixture was first heated. When a mixture is
injected into the column it will either condense onto the stationary phase, dissolve in the liquid on
the stationary phase or remain in the gas phase. If the compound has boiling point that is higher
than the temperature of the column it will condense at the start. Depending on their solubility,
chemicals from the mixture may also be dissolved into the liquid stationary phase. The detector
oven contains a detector that is used to measure which compounds and the quantities of those
compounds that are leaving the column. An example of a detector that could be used is a flame
ionisation detector; this works by burning the organic compounds to produce small amounts of ions
and electrons. The positive ions will be attracted to the cathode and the negative electrons are
attracted to the anode. The loss and gain of electrons from the anode and cathode, when electrons
are neutralised, causes and electrical current which can be measured. The electrical current is
measured by a series of peaks, where each one represents a different compound. The area under
the peaks is proportional to the amount of each compound produced.
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