Separate to identify
Chromatography is known to be a separation technique where it is used to separate
components in a mixture to identify its components. There are many ways chromatography can be
carried out, using paper, a TLC plate as well as gas chromatography. However, this investigation
takes the use of paper and a TLC plate to separate plant pigments as well as an unknown mixture of
amino acids.
This separation technique is used in multitudes of ways such as in pharmaceutical companies
—where the preparation of large quantities of pure materials are needed. Chromatography is also
needed to analyse pure compounds to check for any contamination.
Using the chromatography technique—specifically paper and thin layer chromatography
(TLC) we can separate and analyse how plant pigments separate as a consequence of the different
solubilities. In addition, we can use this technique to find the different components of an unknown
mixture of amino acids.
Apparatus and material:
Grass
Capillary tube
Hairdryer
Pestle and mortar
Sand and solvent
Paper chromatogram
TLC plate
Shandon tank
Beaker
Gas jar
Watch glass
Amino acids
Amino acid mixture X
Ninhydrin dye
Gloves
Method of chromatography of plant pigment on paper
Apparatus and material:
Capillary tube
Grass
Hairdryer
Paper chromatogram
Pestle and mortar
Gas Jar
Gloves
Paper chromatography is the most common chromatography to separate a mixture using the
different solubilities of each component. The components separate in accordance to whether they are
attracted to the stationary or mobile phase.
, The method of chromatography of plant pigment whether on paper or a TLC plate cannot take
place when the plant is a solid—to overcome this, a pestle and mortar was used to grind the grass until
it became a liquid.
1. Using a pestle and mortar, the grass was grinded for a lengthy period.
a. It was grinded with a small amount of isopropyl alcohol which acted as a
solvent as well as sand.
2. When the grass turned into a green liquid, a capillary tube was used to transfer a spot
of plant pigment onto the base line of the paper chromatogram.
a. The base line was drawn onto the chromatogram using pencil as pencil lead
2cm away from the bottom of the paper—pencil lead is not soluble and will
not affect the results of chromatography.
b. The spot of pigment was small and concentrated to offer reliable results.
c. The thumb was pressed onto the end of the capillary tube whilst spotting the
plant pigment so as to maintain the concentration of the plant pigment.
3. A hairdryer was then used to dry the spot of plant pigment before another spot was
added.
a. This process was repeated five times.
4. Using an empty gas jar, the paper chromatogram was placed inside and the base line
was marked on the gas jar using a marker.
a. This line was used as a reference to where the solvent (cyclohexane, propane,
and petroleum in the ratio of 3:2:1) should be filled up to.
5. The paper chromatogram was placed into the solvent in the gas jar.
a. A watch glass was put on top of the gas jar to ensure the paper chromatogram
stood up straight, this was vital so the paper would not absorb more solvent
on one side than the other.
b. In addition, the use of a watch glass was vital so the internal environment of
the gas jar is saturated with the solvent and so there is no extra air flow that
may allow the vapour of the solvent to reach extra parts of the paper, causing
the spot to rise at an angle due to an uneven distribution of the solvent.
6. Once the plant pigment had reached a certain point, the solvent front was marked
with a pencil line and the RF value was calculated.
However, at seeing the results of the paper chromatogram it was clear the plant pigment did
not separate properly. Instead, a TLC plate with a side of silica and plastic which was used to separate
the plant pigment:
Method of chromatography of plant pigment of TLC plate
Thin layer chromatography was used to separate the plant pigments as it offers a better
separation in comparison to paper chromatography. The method of using the TLC plate to separate the
components within a plant pigment follows the same steps as the paper chromatogram. However, the
capillary tube is used to spot the silica side of the TLC plate rather than the plastic side—this was
done gently so not to damage the silica side. It was then placed within a beaker to then go onto
separate the plant pigments.
Method of chromatography for amino acids:
Apparatus and material:
Shandon Tank
Capillary tube
Hairdryer
Amino acids: glycine, asparagine, cystine, leucine and amino acids x
Chromatography is known to be a separation technique where it is used to separate
components in a mixture to identify its components. There are many ways chromatography can be
carried out, using paper, a TLC plate as well as gas chromatography. However, this investigation
takes the use of paper and a TLC plate to separate plant pigments as well as an unknown mixture of
amino acids.
This separation technique is used in multitudes of ways such as in pharmaceutical companies
—where the preparation of large quantities of pure materials are needed. Chromatography is also
needed to analyse pure compounds to check for any contamination.
Using the chromatography technique—specifically paper and thin layer chromatography
(TLC) we can separate and analyse how plant pigments separate as a consequence of the different
solubilities. In addition, we can use this technique to find the different components of an unknown
mixture of amino acids.
Apparatus and material:
Grass
Capillary tube
Hairdryer
Pestle and mortar
Sand and solvent
Paper chromatogram
TLC plate
Shandon tank
Beaker
Gas jar
Watch glass
Amino acids
Amino acid mixture X
Ninhydrin dye
Gloves
Method of chromatography of plant pigment on paper
Apparatus and material:
Capillary tube
Grass
Hairdryer
Paper chromatogram
Pestle and mortar
Gas Jar
Gloves
Paper chromatography is the most common chromatography to separate a mixture using the
different solubilities of each component. The components separate in accordance to whether they are
attracted to the stationary or mobile phase.
, The method of chromatography of plant pigment whether on paper or a TLC plate cannot take
place when the plant is a solid—to overcome this, a pestle and mortar was used to grind the grass until
it became a liquid.
1. Using a pestle and mortar, the grass was grinded for a lengthy period.
a. It was grinded with a small amount of isopropyl alcohol which acted as a
solvent as well as sand.
2. When the grass turned into a green liquid, a capillary tube was used to transfer a spot
of plant pigment onto the base line of the paper chromatogram.
a. The base line was drawn onto the chromatogram using pencil as pencil lead
2cm away from the bottom of the paper—pencil lead is not soluble and will
not affect the results of chromatography.
b. The spot of pigment was small and concentrated to offer reliable results.
c. The thumb was pressed onto the end of the capillary tube whilst spotting the
plant pigment so as to maintain the concentration of the plant pigment.
3. A hairdryer was then used to dry the spot of plant pigment before another spot was
added.
a. This process was repeated five times.
4. Using an empty gas jar, the paper chromatogram was placed inside and the base line
was marked on the gas jar using a marker.
a. This line was used as a reference to where the solvent (cyclohexane, propane,
and petroleum in the ratio of 3:2:1) should be filled up to.
5. The paper chromatogram was placed into the solvent in the gas jar.
a. A watch glass was put on top of the gas jar to ensure the paper chromatogram
stood up straight, this was vital so the paper would not absorb more solvent
on one side than the other.
b. In addition, the use of a watch glass was vital so the internal environment of
the gas jar is saturated with the solvent and so there is no extra air flow that
may allow the vapour of the solvent to reach extra parts of the paper, causing
the spot to rise at an angle due to an uneven distribution of the solvent.
6. Once the plant pigment had reached a certain point, the solvent front was marked
with a pencil line and the RF value was calculated.
However, at seeing the results of the paper chromatogram it was clear the plant pigment did
not separate properly. Instead, a TLC plate with a side of silica and plastic which was used to separate
the plant pigment:
Method of chromatography of plant pigment of TLC plate
Thin layer chromatography was used to separate the plant pigments as it offers a better
separation in comparison to paper chromatography. The method of using the TLC plate to separate the
components within a plant pigment follows the same steps as the paper chromatogram. However, the
capillary tube is used to spot the silica side of the TLC plate rather than the plastic side—this was
done gently so not to damage the silica side. It was then placed within a beaker to then go onto
separate the plant pigments.
Method of chromatography for amino acids:
Apparatus and material:
Shandon Tank
Capillary tube
Hairdryer
Amino acids: glycine, asparagine, cystine, leucine and amino acids x
