Chromatography
33.1 Chromatography:
- Chromatography is a separation technique, where substances are separated by being 33.1 Chromatography
dissolved in a solvent and moved over a solid.
- The mobile phase carries the soluble components with it, the more soluble the faster it moves.
- The stationary phase holds back the components of the mixture that are attracted to it. The more
affinity a component in a mixture has for the stationary phase, the slower it moves with the solvent.
Thin-Layer Chromatography:
- Amino acids can be separated and identified by thin-layer chromatography (TLC).
- The paper is replaced by a thin plastic sheet called the chromatography plate coated in SiO2
(silica/silicon dioxide).
- The solvent is the mobile phase, the powder is the stationary stage
- TLC is better than paper chromatography because it is faster, smaller amounts of mixtures can be
separated, the spots spread out less and the plates are more robust than paper
- Method:
o Spot of amino acid placed on a line 1cm up the plate and placed in a tank with a solvent
0.5cm deep.
o Lid placed on tank to contain the solvent vapour. Solvent rises up the plate carrying amino
acids with it, each lags a different amount depending on its affinity for the solvent compared
to its affinity to the stationary phase.
o The stronger the intermolecular forces between the amino acid and the solvent, the closer the
amino acid is to the solvent.
o When the solvent has nearly reached the top of the plate, the plate is removed from the tank
and the position that the solvent has moved to is marked.
o Because amino acids are colourless, the plate must be sprayed with developing agents (e.g.,
ninhydrin) which react with amino acids to form a purple compound to be able to see the
position they have moved to. UV light can also be used.
o Rf values are calculated for each amino acid.
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o The amino acids are the identified by comparing the Rf value with values of known pure amino
acids run in the same solvent.
- Two-dimensional TLC:
o If two amino acids have very similar Rf values a square plate can be used.
o The plate is spotted in a corner and is used normally. Then the plate is rotated 90o and is ran
again with a different solvent.
o This makes it easier to identify and gives two Rf values,
Column Chromatography:
- Column chromatography uses a powder or resin as the stationary phase e.g., silica or aluminium
- This is put into a narrow tube and a solvent is added at the top, as it runs down the column the
components move at different rates and collect in different flasks at the bottom.
- More than one solvent can be used for better separation.
- Large amounts can be separated and collected
Gas Chromatography:
- The stationary phase is a powder coated in oil, it is coated onto the inside of a long capillary tube and
placed in an oven.
- The mobile phase is an unreactive gas e.g., helium or nitrogen
- The sample is injected and then is carried along by the gas and the mixture separates as some move
along the tube and some are contained by the oil. This means that the components leave the column at
different times (called a retention time)
33.1 Chromatography:
- Chromatography is a separation technique, where substances are separated by being 33.1 Chromatography
dissolved in a solvent and moved over a solid.
- The mobile phase carries the soluble components with it, the more soluble the faster it moves.
- The stationary phase holds back the components of the mixture that are attracted to it. The more
affinity a component in a mixture has for the stationary phase, the slower it moves with the solvent.
Thin-Layer Chromatography:
- Amino acids can be separated and identified by thin-layer chromatography (TLC).
- The paper is replaced by a thin plastic sheet called the chromatography plate coated in SiO2
(silica/silicon dioxide).
- The solvent is the mobile phase, the powder is the stationary stage
- TLC is better than paper chromatography because it is faster, smaller amounts of mixtures can be
separated, the spots spread out less and the plates are more robust than paper
- Method:
o Spot of amino acid placed on a line 1cm up the plate and placed in a tank with a solvent
0.5cm deep.
o Lid placed on tank to contain the solvent vapour. Solvent rises up the plate carrying amino
acids with it, each lags a different amount depending on its affinity for the solvent compared
to its affinity to the stationary phase.
o The stronger the intermolecular forces between the amino acid and the solvent, the closer the
amino acid is to the solvent.
o When the solvent has nearly reached the top of the plate, the plate is removed from the tank
and the position that the solvent has moved to is marked.
o Because amino acids are colourless, the plate must be sprayed with developing agents (e.g.,
ninhydrin) which react with amino acids to form a purple compound to be able to see the
position they have moved to. UV light can also be used.
o Rf values are calculated for each amino acid.
"#$%&'() +,-)" ./ %0) $1,%
- 𝑅! = "#$%&'() +,-)" ./ %0) $,2-)'%
o The amino acids are the identified by comparing the Rf value with values of known pure amino
acids run in the same solvent.
- Two-dimensional TLC:
o If two amino acids have very similar Rf values a square plate can be used.
o The plate is spotted in a corner and is used normally. Then the plate is rotated 90o and is ran
again with a different solvent.
o This makes it easier to identify and gives two Rf values,
Column Chromatography:
- Column chromatography uses a powder or resin as the stationary phase e.g., silica or aluminium
- This is put into a narrow tube and a solvent is added at the top, as it runs down the column the
components move at different rates and collect in different flasks at the bottom.
- More than one solvent can be used for better separation.
- Large amounts can be separated and collected
Gas Chromatography:
- The stationary phase is a powder coated in oil, it is coated onto the inside of a long capillary tube and
placed in an oven.
- The mobile phase is an unreactive gas e.g., helium or nitrogen
- The sample is injected and then is carried along by the gas and the mixture separates as some move
along the tube and some are contained by the oil. This means that the components leave the column at
different times (called a retention time)
