Chromatography
The aim of this practical was to separate the various substances that made up the mixtures that we
used, such as ink, grass pigment and amino acids.
Theory Behind Chromatography:
The primary function of chromatography is to separate and analyse complex mixtures into their
individual components. This technique is widely used in fields such as drug development and forensic
analysis. It is based on the principle of partitioning between a stationary phase and mobile phase. A
stationary phase is a material that stays in a fixed position, while a mobile phase is a fluid or solvent
that moves over the stationary phase, carrying the sample. A solvent is a substance that dissolves a
solute, resulting in a solution.
Chromatography separates molecules based on their interactions with the stationary and mobile
phase. It does separation by molecule size and by polarity interaction. Separation by molecule size
uses a solvent to carry components of a solute based on their solubility, and their ability to bind to the
stationary phase. However, separation by polarity interaction relies on binding interactions between a
protein and another substance. Substances with more polar interactions (stronger attraction to the
polar stationary phase) will move more slowly on the TLC plate or for paper chromatography - this is
because they are more attracted to the surface.
In chromatography, different solvents are used to move the substances along the chromatography
paper. This helps to separate the substances based on how they interact with the solvent, allowing us
to identify and analyse them more effectively.
There are 2 important types of chromatography: HPLC and GC-MS. HPLC (High Performance Liquid
Chromatography) has a liquid mobile phase that carries the sample in a solid material. For example, in
paper chromatography, the solid material is the paper, whilst the liquid mobile phase is the water.
However, in GC-MS (Gas Chromatography-Mass Spectrometry), it involves the separation of volatile
compounds (substances that easily evaporate into the air at room temperature) by gas
chromatography, followed by identification and quantification using mass spectrometry.
Introduction:
Chromatography is a separation technique. This practical is mainly based on the principle of partition.
The molecules inside the mixture applied to the surface used, are separated from each other, and
move up the surface. It consists of 2 phases: Stationary Phase and Mobile Phase. In paper
chromatography, the stationary phase is the paper, and the mobile phase is the solvent.
There are two chromatography techniques that we have learnt. These are: thin-layer chromatography
and paper chromatography. They are both separation techniques, used to separate substances in a
mixture. The main difference between them is the type of material used as the stationary phase. In
TLC, a thin layer of an absorbent material is used, while in paper chromatography, a strip of paper acts
as the stationary phase.
, Method: Amino Acid Chromatography
Apparatus:
• Filter paper
• Pencil
• Tall Beaker
• Paper clip
• Ruler
• Chromatography Solvent
• Ninhydrin
We began by taking some filter paper and a line was drawn in pencil, 5cm from the bottom. This is
called the origin line. 4 crosses were marked in each centre of the origin line and labelled with the
names of each amino acid. A ratio of 25:75 of chromatography solvent (propanone and petroleum
ether) was added into the beaker.
A small amount of each amino acid was spotted on each of their designated crosses on the filter paper
using a capillary tube. After, whilst wearing gloves, the paper was rolled up into a cylinder and secured
with a paper clip. This was then placed inside the tall beaker and a lid was placed on top.
After some time, the solvent slowly moved up the paper by capillary action. Before the solvent had
reached the top, the paper was removed from the beaker and another line was drawn, indicating
where the solvent had stopped. This is called the solvent front. Once the solvent had evaporated, the
paper was hung in a fume cupboard and gently sprayed with some ninhydrin, a developing agent. The
paper was left to dry in the cupboard, giving it time for the spots to develop. After that, the retention
factor, also known as the Rf value, was calculated.
How to calculate the rf value: distance travelled by sample/distance travelled by solvent
Results:
Distance travelled by solvent: 4.9cm
Rf Values
Proline Lysine Asparagine (Unknown)
0.43 0.80 0.39 0.78
Proline: 2.1/4.9 = 0.43
Lysine: 3.9/4.9 = 0.8
Asparagine: 1.9/4.9 = 0.39
(Unknown): 3.8/4.9 = 0.78
Pigment Colour Rf Value
Leucine (Unknown) Yellow 0.75
Lysine Red 0.1764
Proline Pink 0.42857
The aim of this practical was to separate the various substances that made up the mixtures that we
used, such as ink, grass pigment and amino acids.
Theory Behind Chromatography:
The primary function of chromatography is to separate and analyse complex mixtures into their
individual components. This technique is widely used in fields such as drug development and forensic
analysis. It is based on the principle of partitioning between a stationary phase and mobile phase. A
stationary phase is a material that stays in a fixed position, while a mobile phase is a fluid or solvent
that moves over the stationary phase, carrying the sample. A solvent is a substance that dissolves a
solute, resulting in a solution.
Chromatography separates molecules based on their interactions with the stationary and mobile
phase. It does separation by molecule size and by polarity interaction. Separation by molecule size
uses a solvent to carry components of a solute based on their solubility, and their ability to bind to the
stationary phase. However, separation by polarity interaction relies on binding interactions between a
protein and another substance. Substances with more polar interactions (stronger attraction to the
polar stationary phase) will move more slowly on the TLC plate or for paper chromatography - this is
because they are more attracted to the surface.
In chromatography, different solvents are used to move the substances along the chromatography
paper. This helps to separate the substances based on how they interact with the solvent, allowing us
to identify and analyse them more effectively.
There are 2 important types of chromatography: HPLC and GC-MS. HPLC (High Performance Liquid
Chromatography) has a liquid mobile phase that carries the sample in a solid material. For example, in
paper chromatography, the solid material is the paper, whilst the liquid mobile phase is the water.
However, in GC-MS (Gas Chromatography-Mass Spectrometry), it involves the separation of volatile
compounds (substances that easily evaporate into the air at room temperature) by gas
chromatography, followed by identification and quantification using mass spectrometry.
Introduction:
Chromatography is a separation technique. This practical is mainly based on the principle of partition.
The molecules inside the mixture applied to the surface used, are separated from each other, and
move up the surface. It consists of 2 phases: Stationary Phase and Mobile Phase. In paper
chromatography, the stationary phase is the paper, and the mobile phase is the solvent.
There are two chromatography techniques that we have learnt. These are: thin-layer chromatography
and paper chromatography. They are both separation techniques, used to separate substances in a
mixture. The main difference between them is the type of material used as the stationary phase. In
TLC, a thin layer of an absorbent material is used, while in paper chromatography, a strip of paper acts
as the stationary phase.
, Method: Amino Acid Chromatography
Apparatus:
• Filter paper
• Pencil
• Tall Beaker
• Paper clip
• Ruler
• Chromatography Solvent
• Ninhydrin
We began by taking some filter paper and a line was drawn in pencil, 5cm from the bottom. This is
called the origin line. 4 crosses were marked in each centre of the origin line and labelled with the
names of each amino acid. A ratio of 25:75 of chromatography solvent (propanone and petroleum
ether) was added into the beaker.
A small amount of each amino acid was spotted on each of their designated crosses on the filter paper
using a capillary tube. After, whilst wearing gloves, the paper was rolled up into a cylinder and secured
with a paper clip. This was then placed inside the tall beaker and a lid was placed on top.
After some time, the solvent slowly moved up the paper by capillary action. Before the solvent had
reached the top, the paper was removed from the beaker and another line was drawn, indicating
where the solvent had stopped. This is called the solvent front. Once the solvent had evaporated, the
paper was hung in a fume cupboard and gently sprayed with some ninhydrin, a developing agent. The
paper was left to dry in the cupboard, giving it time for the spots to develop. After that, the retention
factor, also known as the Rf value, was calculated.
How to calculate the rf value: distance travelled by sample/distance travelled by solvent
Results:
Distance travelled by solvent: 4.9cm
Rf Values
Proline Lysine Asparagine (Unknown)
0.43 0.80 0.39 0.78
Proline: 2.1/4.9 = 0.43
Lysine: 3.9/4.9 = 0.8
Asparagine: 1.9/4.9 = 0.39
(Unknown): 3.8/4.9 = 0.78
Pigment Colour Rf Value
Leucine (Unknown) Yellow 0.75
Lysine Red 0.1764
Proline Pink 0.42857
