Chromatography
Chromatography is used to separate substances in a mixture.
There are two phases:
- A mobile phase - where molecules can move (always a liquid/gas)
- A stationary phase - where molecules can’t move (a solid or liquid on a solid support)
A mobile phase moves through or over the stationary phase.
The distance each substance moves depends on its solubility in the mobile phase and its
retention by the stationary phase.
The more soluble the component in the mobile phase, the further it will travel than
components that are more strongly adsorbed (attracted) to the stationary phase (high
retention).
The differences in solubility and retention separates out the different substances.
TLC PRACTICAL 12
The mobile phase is a liquid solvent (ethanol).
The stationary phase is a thin layer of silica/alumina fixed to a glass/metal plate.
1. Draw a line in pencil near the bottom of the TLC plate. This is the baseline. Put a very
small drop of each mixture on the line using a capillary tube.
2. Allow the spots on the plate to dry.
3. Place the plate in a beaker with a small volume of solvent. This is the mobile phase.
The solvent level must be below the baseline so it doesn’t dissolve the samples away.
Cover the beaker with a glass lid.
4. The solvent will start to move up the plate. As it moves it will carry the substances in
the mixture with it. Chemicals that are carried faster will travel further up the plate.
5. Leave the beaker until the solvent moves almost to the top of the plate. Then remove
the plate from the beaker.
6. Before it’s evaporated use a pencil to mark how far the solvent travelled up the plate.
This is the solvent front
7. Place the plate in a fume cupboard and leave it to dry. The fume cupboard will
prevent any toxic or flammable fumes from escaping into the room.
8. The result is a chromatogram and you can use the positions of the chemicals on the
chromatogram to identify what the chemicals are.
, If the chemicals in the mixture are colourless (amino acids) then you need to make them
visible.
- Fluorescent dye is added to the silica/alumina layer that glows when UV light shines
on it. You can put the plate under a UV lamp and draw around the dark patches ro
show where the spots of chemical are.
- Expose the chromatogram to iodine vapour by leaving the plate in a sealed jar with a
couple of iodine crystals. The iodine vapour is a locating agent so it sticks to
chemicals on the plate and they show up as purple spots.
Rf values are used to find out what each chemical is.
Rf =
When measuring the Rf value, measure from baseline to vertical centre of the spot and from
baseline to solvent front.
Rf values don’t depend on how big the plate is or how far the solvent trave;s.
The Rf value is a property of a chemical in the mixture and so can be used to identify them
by looking the Rf value up in a table of standard Rf values.
If the composition of the TLC plate, the solvent or the temperature at which you carry out
your chromatography experiment changes even slightly you will get different Rf values.
It is hard to keep chromatography conditions identical so if you suspect the mixture contains
a certain chemical, then put a spot of that pure chemical on the baseline of the same plate
and run them both at the same time.
Chromatography is used to separate substances in a mixture.
There are two phases:
- A mobile phase - where molecules can move (always a liquid/gas)
- A stationary phase - where molecules can’t move (a solid or liquid on a solid support)
A mobile phase moves through or over the stationary phase.
The distance each substance moves depends on its solubility in the mobile phase and its
retention by the stationary phase.
The more soluble the component in the mobile phase, the further it will travel than
components that are more strongly adsorbed (attracted) to the stationary phase (high
retention).
The differences in solubility and retention separates out the different substances.
TLC PRACTICAL 12
The mobile phase is a liquid solvent (ethanol).
The stationary phase is a thin layer of silica/alumina fixed to a glass/metal plate.
1. Draw a line in pencil near the bottom of the TLC plate. This is the baseline. Put a very
small drop of each mixture on the line using a capillary tube.
2. Allow the spots on the plate to dry.
3. Place the plate in a beaker with a small volume of solvent. This is the mobile phase.
The solvent level must be below the baseline so it doesn’t dissolve the samples away.
Cover the beaker with a glass lid.
4. The solvent will start to move up the plate. As it moves it will carry the substances in
the mixture with it. Chemicals that are carried faster will travel further up the plate.
5. Leave the beaker until the solvent moves almost to the top of the plate. Then remove
the plate from the beaker.
6. Before it’s evaporated use a pencil to mark how far the solvent travelled up the plate.
This is the solvent front
7. Place the plate in a fume cupboard and leave it to dry. The fume cupboard will
prevent any toxic or flammable fumes from escaping into the room.
8. The result is a chromatogram and you can use the positions of the chemicals on the
chromatogram to identify what the chemicals are.
, If the chemicals in the mixture are colourless (amino acids) then you need to make them
visible.
- Fluorescent dye is added to the silica/alumina layer that glows when UV light shines
on it. You can put the plate under a UV lamp and draw around the dark patches ro
show where the spots of chemical are.
- Expose the chromatogram to iodine vapour by leaving the plate in a sealed jar with a
couple of iodine crystals. The iodine vapour is a locating agent so it sticks to
chemicals on the plate and they show up as purple spots.
Rf values are used to find out what each chemical is.
Rf =
When measuring the Rf value, measure from baseline to vertical centre of the spot and from
baseline to solvent front.
Rf values don’t depend on how big the plate is or how far the solvent trave;s.
The Rf value is a property of a chemical in the mixture and so can be used to identify them
by looking the Rf value up in a table of standard Rf values.
If the composition of the TLC plate, the solvent or the temperature at which you carry out
your chromatography experiment changes even slightly you will get different Rf values.
It is hard to keep chromatography conditions identical so if you suspect the mixture contains
a certain chemical, then put a spot of that pure chemical on the baseline of the same plate
and run them both at the same time.
