Chromatography - an introduction
Chromatography is the process used for separating substances in a mixture. This can be
useful for several investigative purposes, for example making comparisons between inks
and drawing conclusions based on similarities and differences. There are two phases in
chromatography: the mobile phase and the stationary phase. The stationary phase does not
move, for example the absorbent paper used in paper chromatography. The mobile phase is
the solvent that moves through the paper and carries the different substances within a
mixture with it (BBC Bitesize, n.d.). Each substance is attracted to the two phases in different
proportions, which causes them to move at different rates through the paper. The Rf value
(retention factor) tells us whether the substance prefers the stationary or mobile phase, and
can also help us determine polarity and relative masses (BYJUS, n.d.). The Rf value is
calculated by the distance travelled by the spot divided by the distance travelled by the
solvent.
Polar components in mixtures dissolve easily in polar solvents, and non-polar components
dissolve easily in non-polar solvents. The polarity of the solvents that we use increases in
the order:
petroleum ether, diethyl ether, ethyl ethanoate, propanone, ethanol, water
A tilted chromatogram and large spots can be solved by using a limited number of spots
when dotting the pigments at the base line of the TLC plate or chromatography paper as this
ensures that the dots are concentrated and don’t stray from where they are deposited.
Tilted chromatograms are when the chromatogram paper is not sat straight in the beaker
which causes the pigments to merge as they travel up the paper as it always travels up no
matter what way the paper is sitting - this can be prevented by using a splint and a paperclip
to hold the paper upright and straight.
When spotting the pigments at the base line of the chromatography paper, we can stretch
out the capillary tube using a bunsen burner in order to make it thinner and create smaller
spots. As well as this, when dotting them we can wait for it to dry and then dot it again -
when this is repeated several times, it means that the dot is in high concentration to create a
good chromatogram, without the spot spreading at the base line and colliding with the other
pigments when they are travelling with the solvent.
Health and safety
- Gloves must be worn as solvents are harmful when absorbed through the skin
- Recommended to be carried out in a fume cupboard as inhaling the fumes too much
can be damaging
Leaf pigments - paper chromatography
Method
1. Grind a few leaves of your choice with sand in a mortar with a pestle. Use about 20
cm3 of propanone. This extracts a considerable amount of pigment.
2. Filter the mixture through filter paper in a filter funnel into a sample bottle. Add a little
more solvent if it looks like the solvent has evaporated. (Ask the technician or tutor if
you need to evaporate the solvent to make the solution more concentrated.)
3. Obtain three similarly sized pieces of chromatography paper. Put your initials on the
top right-hand corners faintly in pencil.
4. Draw a faint pencil line 1.5 cm from the bottom of each plate – above the level that
the solvent will reach (have a look at the solvent tanks in the fume cupboard). Check
that 1.5 cm is sufficient. Cross your line with a faint pencil line in the middle of the
original line.
, 5. Using a glass spotting tube, spot the pigment solution on to the spot and allow it to
dry.
6. Re-spot with the solution and allow it to dry. Repeat this at least 20 times to get a
really dark, concentrated, small spot.
7. Repeat steps 4, 5 and 6 for the other two plates.
8. Place one plate into 100% petroleum ether (least polar), one into 100% propanone
(most polar) and one into a 70:30 mixture of petroleum ether and propanone (polarity
between the other two). The solvents are in tanks in the fume cupboard.
9. Once the solvent fronts have nearly reached the tops of the plates, remove them
from the solvents. Mark the position of the solvent fronts with pencil lines in the fume
cupboard.
10. Once the plates are dry, take them back to your bench and sketch the results.
Results
Unable to calculate Rf values from these chromatograms
Conclusion
This practical was not suitable for separating the leaf pigments as shown in the results which
are insufficient - this result was seen throughout the class meaning that it wasn’t a problem
with the method or human error, the practical was just not suitable for producing valid data.
The pigments didn’t stick to the paper very well which is why the solvent completely carried
or didn’t carry the pigments at all depending on their polarity. Petroleum ether is less polar
than propanone, so we can infer that the leaf pigments were polar as they were carried by
the propanone. They didn’t separate due to their inability to stick to the paper, so all of the
separated pigments were carried completely with the solvent and travelled at the same rate.
Larger molecules take longer to move up the chromatography paper, whilst smaller
molecules are more mobile. It’s impossible to determine the size of any molecules in the leaf
pigment in these chromatograms as they’re all too close together to tell one another apart.
An improvement to this method would just be to use TLC instead of paper chromatography,
as the pigments would stick to the paper better and we would be able to view the separation
of the pigments enough to calculate the Rf values and assess the polarity, size of molecules,
and compare the pigments efficiently.
Chromatography is the process used for separating substances in a mixture. This can be
useful for several investigative purposes, for example making comparisons between inks
and drawing conclusions based on similarities and differences. There are two phases in
chromatography: the mobile phase and the stationary phase. The stationary phase does not
move, for example the absorbent paper used in paper chromatography. The mobile phase is
the solvent that moves through the paper and carries the different substances within a
mixture with it (BBC Bitesize, n.d.). Each substance is attracted to the two phases in different
proportions, which causes them to move at different rates through the paper. The Rf value
(retention factor) tells us whether the substance prefers the stationary or mobile phase, and
can also help us determine polarity and relative masses (BYJUS, n.d.). The Rf value is
calculated by the distance travelled by the spot divided by the distance travelled by the
solvent.
Polar components in mixtures dissolve easily in polar solvents, and non-polar components
dissolve easily in non-polar solvents. The polarity of the solvents that we use increases in
the order:
petroleum ether, diethyl ether, ethyl ethanoate, propanone, ethanol, water
A tilted chromatogram and large spots can be solved by using a limited number of spots
when dotting the pigments at the base line of the TLC plate or chromatography paper as this
ensures that the dots are concentrated and don’t stray from where they are deposited.
Tilted chromatograms are when the chromatogram paper is not sat straight in the beaker
which causes the pigments to merge as they travel up the paper as it always travels up no
matter what way the paper is sitting - this can be prevented by using a splint and a paperclip
to hold the paper upright and straight.
When spotting the pigments at the base line of the chromatography paper, we can stretch
out the capillary tube using a bunsen burner in order to make it thinner and create smaller
spots. As well as this, when dotting them we can wait for it to dry and then dot it again -
when this is repeated several times, it means that the dot is in high concentration to create a
good chromatogram, without the spot spreading at the base line and colliding with the other
pigments when they are travelling with the solvent.
Health and safety
- Gloves must be worn as solvents are harmful when absorbed through the skin
- Recommended to be carried out in a fume cupboard as inhaling the fumes too much
can be damaging
Leaf pigments - paper chromatography
Method
1. Grind a few leaves of your choice with sand in a mortar with a pestle. Use about 20
cm3 of propanone. This extracts a considerable amount of pigment.
2. Filter the mixture through filter paper in a filter funnel into a sample bottle. Add a little
more solvent if it looks like the solvent has evaporated. (Ask the technician or tutor if
you need to evaporate the solvent to make the solution more concentrated.)
3. Obtain three similarly sized pieces of chromatography paper. Put your initials on the
top right-hand corners faintly in pencil.
4. Draw a faint pencil line 1.5 cm from the bottom of each plate – above the level that
the solvent will reach (have a look at the solvent tanks in the fume cupboard). Check
that 1.5 cm is sufficient. Cross your line with a faint pencil line in the middle of the
original line.
, 5. Using a glass spotting tube, spot the pigment solution on to the spot and allow it to
dry.
6. Re-spot with the solution and allow it to dry. Repeat this at least 20 times to get a
really dark, concentrated, small spot.
7. Repeat steps 4, 5 and 6 for the other two plates.
8. Place one plate into 100% petroleum ether (least polar), one into 100% propanone
(most polar) and one into a 70:30 mixture of petroleum ether and propanone (polarity
between the other two). The solvents are in tanks in the fume cupboard.
9. Once the solvent fronts have nearly reached the tops of the plates, remove them
from the solvents. Mark the position of the solvent fronts with pencil lines in the fume
cupboard.
10. Once the plates are dry, take them back to your bench and sketch the results.
Results
Unable to calculate Rf values from these chromatograms
Conclusion
This practical was not suitable for separating the leaf pigments as shown in the results which
are insufficient - this result was seen throughout the class meaning that it wasn’t a problem
with the method or human error, the practical was just not suitable for producing valid data.
The pigments didn’t stick to the paper very well which is why the solvent completely carried
or didn’t carry the pigments at all depending on their polarity. Petroleum ether is less polar
than propanone, so we can infer that the leaf pigments were polar as they were carried by
the propanone. They didn’t separate due to their inability to stick to the paper, so all of the
separated pigments were carried completely with the solvent and travelled at the same rate.
Larger molecules take longer to move up the chromatography paper, whilst smaller
molecules are more mobile. It’s impossible to determine the size of any molecules in the leaf
pigment in these chromatograms as they’re all too close together to tell one another apart.
An improvement to this method would just be to use TLC instead of paper chromatography,
as the pigments would stick to the paper better and we would be able to view the separation
of the pigments enough to calculate the Rf values and assess the polarity, size of molecules,
and compare the pigments efficiently.
