Mandatory Experiment 7.7
Separation of a Mixture of Indicators by Chromatographic
Techniques
Student Material
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:
Method
Stationary phase
Moving phase
Paper
chromatography
Thin layer
chromatography
Column
chromatography
Water on paper
Liquid
Solid (e.g. silica
gel)
Solid
Liquid
Retention
mechanism
Partition (between
two solvents)
Adsorption
Liquid
Adsorption
The column chromatography experiment involves forcing the mixture of indicators
under pressure through a solid phase extraction column. A very rapid separation of the
components occurs, and the different components of the mixture can be collected in
separate containers. The components can be tentatively identified by observing their
colours in acid and in base.
Results obtained using paper chromatography or thin layer chromatography can be
recorded in terms of Rf values. The Rf value for a component of a mixture is equal to
Distance travelled by the component

Distance travelled by the solvent front
Rf values may be used to tentatively identify the components of a mixture of
indicators.
1
(a) Separation of a mixture of indicators using paper
chromatography
Chemicals and Apparatus
Universal Indicator solution
(or other mixture of indicators)
Methyl orange solution
Bromothymol blue solution
Phenolphthalein solution
Ethanol
Deionised water
0.88 ammonia solution
Dilute ammonia solution
Water/ethanol/ammonia solution solvent (5:2:1)
Capillary tubes
Paper chromatography tank
Solvent trough for descending chromatography
Chromatography paper
Hair drier
Procedure
NB: Wear your safety glasses.
2
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.
5. Remove and dry.
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.)
7. Identify the indicators present in the mixture.
Results
Distance travelled by solvent front
Distance travelled by methyl orange
Rf value
Distance travelled by phenolphthalein
Rf value
Distance travelled by bromothymol blue
Rf value
Distance travelled by fastest moving component of mixture
Rf value
Distance travelled by next fastest moving component of mixture
Rf value
Distance travelled by next fastest moving component of mixture
Rf value
Distance travelled by next fastest moving component of mixture
Rf value
Indicators present in mixture
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
Questions relating to the experiment
1. Why is a paper chromatography tank not used for a considerable time after the
chromatography solvent has been added?
2. Why are two lines usually drawn on a paper chromatogram?
3
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?
4
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

Descending paper chromatography is an alternative to the method described. The
trough is half-filled with the water/ethanol/ammonia solution solvent, and the
covered tank allowed to stand for a few hours.

In this method, the paper dips into the solvent in the trough. The solvent moves
down the paper during the experiment.

Ascending paper chromatography can be carried out in a covered beaker, although
better results are achievable using a tank.

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.
5

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

The solvent for paper chromatography is prepared as follows: In a fume
cupboard, pour 100 cm3 of ethanol, 250 cm3 of water and 50 cm3 of 0.88
ammonia solution into a large beaker and mix well.

An alternative solvent for the paper chromatography experiment is
water/ethanol/saturated ammonium sulphate solution (5:2:1).

Dilute ammonia solution (approximately 1M) is prepared as follows: Using a
fume cupboard, measure out 57 cm3 of concentrated ammonia solution in a
graduated cylinder. Add this to about 500 cm3 of deionised water in a beaker,
and stir. Pour the solution into a 1 litre graduated cylinder. Dilute to 1 litre
with water. Add to a labelled bottle and mix well.
Safety considerations



Safety glasses must be worn.
The use of gloves is recommended.
The solutions should be made up in a fume cupboard.
Chemical hazard notes
Concentrated ammonia solution
irritating to the lungs.
Ethanol
can cause burns. Ammonia vapour is toxic and
is volatile and flammable.
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.
6
Disposal of wastes
Dilute with water, and flush to foul water drain.
Specimen results and calculations
Distance travelled by solvent front
Distance travelled by methyl orange
Rf value
Distance travelled by phenolphthalein
Rf value
Distance travelled by bromothymol blue
Rf value
Distance travelled by fastest moving component of mixture
Rf value
Distance travelled by next fastest moving component of mixture
Rf value
Distance travelled by next fastest moving component of mixture
Rf value
Distance travelled by next fastest moving component of mixture
Rf value
Indicators present in mixture
blue, methyl orange, phenolphthalein and one other (at least)
= 10 cm
= 6.8 cm
= 0.68
= 8.4 cm
= 0.84
= 9.7 cm
= 0.97
= 9.7 cm
= 0.97
= 8.4 cm
= 0.84
= 7.9 cm
= 0.79
= 6.8 cm
= 0.68
= bromothymol
Suggested solutions to student questions
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.
2. Why are two lines usually drawn on a paper chromatogram?
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.
3. When is it possible to separate two components of a mixture using paper
chromatography?
When one of the components is attracted to a significantly different extent by the
stationary phase and/or the mobile phase.
4. When two substances are found to have different Rf values in an experiment
carried out under the same conditions, what does this mean?
The two substances are not identical.
7
(b) Separation of a mixture of indicators using thin layer
chromatography
Student Material
Chemicals and Apparatus
Universal Indicator solution
Methyl orange solution
Bromothymol blue solution
Phenolphthalein solution
Industrial methylated spirits
Dilute ammonia solution
Capillary tubes
Thin layer chromatography tank
Thin layer plates (aluminium or plastic backed)
Hair drier
Procedure
NB: Wear your safety glasses.
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.
3. Cover the tank, and allow to stand for 10 minutes.
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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.
9. Calculate and record the Rf values of each indicator.
10. Identify the indicators present in the mixture.
Results
Distance travelled by solvent front
Distance travelled by methyl orange
Rf value
Distance travelled by phenolphthalein
Rf value
Distance travelled by bromothymol blue
Rf value
Distance travelled by fastest moving component of mixture
Rf value
Distance travelled by next fastest moving component of mixture
Rf value
Distance travelled by next fastest moving component of mixture
Rf value
Distance travelled by next fastest moving component of mixture
Rf value
Indicators present in mixture
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
Questions relating to the experiment
1. Why is filter paper placed around the walls of a thin layer chromatography tank?
2. Why are two lines usually drawn on a thin layer chromatogram?
3. When is it possible to separate components of a mixture using thin layer
chromatography?
4. When two substances are found to have identical Rf values, what does this mean?
9
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.

Industrial methylated spirits is colourless, and should not be confused with
methylated spirits for domestic use, which contains a dye.

Universal indicator or a mixture of common indicators (e.g. methyl red +
bromothymol blue + methyl orange) may be used in this experiment.

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

Dilute ammonia solution (approximately 1M) is prepared as follows: Using a
fume cupboard, measure out 57 cm3 of concentrated ammonia solution in a
graduated cylinder. Add this to about 500 cm3 of deionised water in a beaker,
and stir. Pour the solution into a 1 litre graduated cylinder. Dilute to 1 litre
with water. Add to a labelled bottle and mix well.
Safety considerations



Safety glasses must be worn.
The use of gloves is recommended.
The ammonia solution should be made up in a fume cupboard.
Chemical hazard notes
Concentrated ammonia solution
irritating to the lungs.
can cause burns. Ammonia vapour is toxic and
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Methylated spirits
is volatile and flammable.
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
Dilute with water, and flush to foul water drain.
Specimen results and calculations
Distance travelled by solvent front
= 6.8 cm
Distance travelled by methyl orange
= 3.9 cm
Rf value
= 0.57
Distance travelled by phenolphthalein
= 4.5 cm
Rf value
= 0.66
Distance travelled by bromothymol blue
= 4.7 cm
Rf value
= 0.69
Distance travelled by fastest moving component of mixture
= 4.7 cm
Rf value
= 0.69
Distance travelled by next fastest moving component of mixture = 3.9 cm
Rf value
= 0.57
Distance travelled by next fastest moving component of mixture = 3.7 cm
Rf value
= 0.54
Indicators present in mixture
= methyl orange,
bromothymol blue, and one other (at least) – phenolphthalein not identified
Suggested answers to student questions
1. Why is filter paper placed around the walls of a thin layer chromatography tank?
To speed up saturation of the tank with solvent vapour.
2. Why are two lines usually drawn on a thin layer chromatogram?
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.
3. When is it possible to separate components of a mixture using thin layer
chromatography?
When one of the components is attracted to a significantly different extent by the
stationary phase and/or the mobile phase.
11
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.
12
(c) Separation of a mixture of indicators using column
chromatography
Student Material
Chemicals and Apparatus
Universal Indicator solution
Methyl red solution
Methyl orange solution
Bromothymol blue solution
Phenolphthalein solution
Methanol
35% methanol solution
60% methanol solution
Dilute sodium hydroxide solution
Plastic disposable syringe
Solid phase extraction column
Adaptor (to connect syringe to column)
Small test tubes and test tube rack
Procedure
NB: Wear your safety glasses and gloves.
13
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.
7. Using the plunger force the mixture through the column.
8. Collect the different components of the mixture in separate test tubes.
9. Describe the colour of each of the components.
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.
Questions relating to the experiment
1. When is it possible to separate components of a mixture using column
chromatography?
2. What is the purpose of the syringe when components of a mixture are being
separated using a solid phase extraction column?
3. Why is an adaptor necessary?
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?
14
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.

Universal indicator or a mixture of common indicators (e.g. methyl red +
bromothymol blue + phenolphthalein + methyl orange) may be used in this
experiment.

When a sample of universal indicator is forced into the column, a number of
colours are observed on the column, including orange and yellow. This indicates
that separation of the components of the mixture is happening. After the 35%
methanol stage of the experiment, a light purple and a pink component can be
seen. After the 60% methanol stage, a light pink colour remains on the column.

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.
Dilute sodium hydroxide solution (1 M): Carefully add, in stages, 40 g of sodium
hydroxide with constant stirring to about 800 cm3 of water. Continue stirring until all
of the solid has dissolved. Make the solution up to 1 litre. Stopper, and mix
thoroughly.
Safety considerations



Safety glasses must be worn.
The use of gloves is recommended, and they should definitely be worn when
the solutions are being made up.
The methanol solution and the hydrochloric acid solution should be made up
in a fume cupboard.
15
Chemical hazard notes
Methanol
is volatile and flammable. It is also toxic by inhalation, if
swallowed and by skin absorption.
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.
Dilute sodium hydroxide solution
is also corrosive.
Disposal of wastes
Dilute with water, and flush to foul water drain.
Suggested answers to student questions
1. When is it possible to separate components of a mixture using column
chromatography?
When one of the components is attracted to a significantly different extent by the
stationary phase and/or the mobile phase.
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.
3. Why is an adaptor necessary?
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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