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Theory
Chromatography involves the use of a mobile phase and a stationary phase to separate
the components of a mixture from each other. The separation is based on the fact that
different components of a mixture are attracted to different extents to the mobile
phase as compared to the stationary phase. A component that is attracted more to the
stationary phase will move more slowly during the separation than one that is
attracted more by the mobile phase. The chromatographic methods that may be used
in this experiment can be summarised as follows:
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(a) Separation of a mixture of indicators using paper
chromatography
Capillary tubes
Paper chromatography tank
Solvent trough for descending chromatography
Chromatography paper
Hair drier
Procedure
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1. Add solvent to the bottom of the tank to a depth of about 10 mm. Cover the tank,
and allow to stand for a few hours, to allow the tank to become saturated with
solvent vapour.
2. Make a line with a pencil near the top of a rectangular sheet of chromatography
paper, and another line about 3 cm from the bottom.
3. Place a small spot of each indicator and of the mixture of indicators at different
points on the line near the bottom of the paper, using a capillary tube. Dry using a
hair drier, and repeat.
4. Place the chromatogram in the tank, ensuring that the solvent level in the tank is
below the line on which the indicator samples are spotted. Run the chromatogram,
until the solvent reaches the line near the top of the paper.
6. Calculate and record the Rf values of each indicator. (Ammonia vapour is used to
locate phenolphthalein – the paper is held in vapour coming from a boiling
solution of dilute ammonia.)
Results
1. Why is a paper chromatography tank not used for a considerable time after the
chromatography solvent has been added?
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3. When is it possible to separate components of a mixture using paper
chromatography?
4. When two substances are found to have different Rf values, what does this mean?
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Teacher Material
Fine, drawn out capillaries are best for paper chromatography, and they should be
placed in a broken glass bin after use.
To make a fine capillary of this type, hold a suitable length of glass tubing in a
very hot Bunsen flame and continuously rotate it until it begins to soften.
Immediately withdraw it from the flame, pulling gently at both ends to draw it out.
Allow to cool, and then gently break it into two capillaries
In this method, the paper dips into the solvent in the trough. The solvent moves
down the paper during the experiment.
It can take several hours to saturate a paper chromatography tank with solvent
vapour, depending on the size of the tank. It is necessary to saturate the tank in
order to get optimum and reproducible results. The tank should therefore be set up
well in advance of the experiment, perhaps on the previous day.
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Universal indicator or a mixture of common indicators (e.g. methyl red +
bromothymol blue + methyl orange) may be used in this experiment.
There are a number of different universal indicators. One type contains the
following indicators: bromothymol blue, methyl orange, methyl red and
phenolphthalein.
Identical Rf values are not sufficient in themselves to confirm that two samples are
identical. They merely indicate that this may be the case. If it is necessary to
definitively confirm the identity of two samples, other non-chromatographic
techniques have to be used. For the purposes of this experiment, however,
identical Rf values may be taken as a good indication that the samples in question
are the same.
Preparation of reagents
Safety considerations
Concentrated ammonia solution can cause burns. Ammonia vapour is toxic and
irritating to the lungs.
Solutions of methyl orange and other indicators usually contain 50% ethanol and
are flammable. Bromothymol blue solution, which contains approximately 80%
water, is an exception.
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Disposal of wastes
1. Why is a paper chromatography tank not used for a considerable time after the
chromatography solvent has been added?
To allow time for the tank to become saturated with solvent vapour.
One line is needed to indicate where the samples start from, and the other to
indicate the distance travelled by the solvent front, which enables the Rf values to
be calculated.
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(b) Separation of a mixture of indicators using thin layer
chromatography
Student Material
Procedure
1. Cut a piece of filter paper to fit around the walls of the tank.
2. Add enough industrial methylated spirits to the tank to allow it to saturate the
filter paper and give a depth of about 10mm at the bottom.
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4. Draw a line lightly with a pencil on a thin layer plate a little more than 10 mm
from the bottom of the plate. Draw a horizontal line near the top of the plate.
5. Using a capillary tube, place a small spot of each sample on the line near the
bottom of the plate. Allow to dry – a hair drier may be used to speed up drying.
6. Stand the plate carefully in the tank, ensuring that the samples are above the
surface of the liquid. Cover the tank, and allow the solvent front to rise up the
plate to the line near the top. Remove the plate, and allow to dry.
8. Note the colours of the zones. (Ammonia vapour is used to locate phenolphthalein
– the plate is held in vapour coming from a boiling solution of dilute ammonia.)
Draw a diagram of the plate, labelling the zones.
Results
1. Why is filter paper placed around the walls of a thin layer chromatography tank?
4. When two substances are found to have identical Rf values, what does this mean?
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Teacher Material
Fine, drawn out capillaries are best for thin layer chromatography, and they should
be placed in a broken glass bin after use.
To make a fine capillary of this type, continuously rotate a suitable length of glass
tubing in a very hot Bunsen flame until it begins to soften. Immediately withdraw
it from the flame, pulling gently at both ends to draw it out. Allow to cool, and
then gently break it into two capillaries
An empty coffee jar with lid or a beaker with a clock glass as a cover may be used
as an alternative to a thin layer chromatography tank.
Aluminium-backed thin layer plates have a very fine coating of stationary phase,
which may be entirely scraped off when the line is being drawn unless care is
taken.
Identical Rf values are not sufficient in themselves to confirm that two samples are
identical. They merely indicate that this may be the case. If it is necessary to
definitively confirm the identity of two samples, other non-chromatographic
techniques have to be used. For the purposes of this experiment, however,
identical Rf values may be taken as a good indication that the samples in question
are the same.
Preparation of reagents
Safety considerations
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Concentrated ammonia solution can cause burns. Ammonia vapour is toxic and
irritating to the lungs.
Solutions of methyl orange and other indicators usually contain 50% ethanol and
are flammable. Bromothymol blue solution, which contains approximately 80%
water, is an exception.
Disposal of wastes
1. Why is filter paper placed around the walls of a thin layer chromatography tank?
One line is needed to indicate where the samples start from, and the other to
indicate the distance travelled by the solvent front, which enables the Rf values to
be calculated.
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4. When two substances are found to have identical Rf values, what does this mean?
The two substances may be the same, but further evidence is necessary to
establish this conclusively.
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(c) Separation of a mixture of indicators using column
chromatography
Student Material
Procedure
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1. Flush the solid phase extraction column through with methanol, using the plunger.
2. Flush the solid phase extraction column through with water a number of times.
3. Place a sample of the mixture of indicators on the top of the column – the sample
should cover the top of the column to a depth of slightly less than half the length
of the column solid phase.
4. Half fill the syringe with air and, using the adaptor, attach the syringe to the
column. Using the plunger, gently force the mixture into the column.
5. Have several small test tubes available to collect the different components of the
mixture.
6. Add about 4 cm3 of 35% methanol solution to the syringe, and using the adaptor,
attach the syringe to the column.
10. When the liquid emerging from the column is colourless, repeat steps 6 to 9, using
60% methanol instead of 35% methanol.
11. Some of the components can be tentatively identified as follows: Add a little
dilute sodium hydroxide solution to each of the test tubes collected. Record the
colours observed – these may then be used to try and identify the indicator
present.
12. Wash out the solid phase alternately with 100% methanol followed by excess
water until no colour remains in the column.
2. What is the purpose of the syringe when components of a mixture are being
separated using a solid phase extraction column?
4. Why is it necessary to flush a solid phase extraction column with methanol and
then with water before using it to carry out a separation?
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Teacher Material
Solid phase extraction columns vary in size and polarity. There is an element of
trial and error involved in selecting a suitable solvent and column for the
separation of the components of a particular mixture.
The use of an adaptor is essential in order to be able to force the solvent under
pressure through the column. This is important as it allows separations to be
achieved in a matter of minutes.
Solid phase extraction columns are best rinsed after use as follows: Using the
plunger, wash out the column first with 100% methanol followed by excess water.
Continue to wash out the column alternately with 100% methanol followed by
excess water until no colour remains in the column.
Preparation of reagents
35% methanol solution: Using a fume cupboard, dilute 175 cm3 of methanol to 500
cm3 with deionised water. Stopper, and mix thoroughly.
Safety considerations
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Chemical hazard notes
Solutions of methyl orange and other indicators usually contain 50% ethanol and
are flammable. Bromothymol blue solution, which contains approximately 80%
water, is an exception.
Solid sodium hydroxide is corrosive, and can cause severe burns to eyes and
skin. Always wear eye protection.
Disposal of wastes
2. What is the purpose of the syringe when components of a mixture are being
separated using a solid phase extraction column?
To allow the solvent to be forced through the column under pressure, thereby
achieving a rapid separation.
To enable the syringe to be fitted exactly into the column – without this, it is not
possible to force the liquid through the column under pressure.
4. Why is it necessary to flush a solid phase extraction column with methanol and
then with water before using it to carry out a separation?
Methanol will remove any residual organic material from the column. Water will
then remove any remaining methanol.
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