Biology assignment
My name is Marwa and I am a newly appointed technical assistant for Chemicalequip, in this
assignment, I will carry out different chromatographic techniques such as ink paper chromatography,
chromatography of leaf pigments, thin-layer chromatography and paper chromatography of amino
acid. Therefore, I will demonstrate my skill to be able to perform a variety of practical procedures
and techniques for separating and identifying components in a mixture. I will also describe the
method, and types of equipment, analyse and evaluate the results, compare my results with
published results and suggest improvements.
What is chromatography?
Mikhail Tswett discovered chromatography during his studies on plant pigment in 1901. He applied
the method and the technique on how to separate plant pigment including chlorophyll, xanthophyll,
and carotenoid. Chromatography is a laboratory technique that is used to separate a mixture of
different components such as ink chromatography which is used to analyse the mixture by separating
the ink into what they are made of, which means that each component of the mixture will move at
different rates as the paper is placed inside the beaker. This will cause the mixture to separate out
into coloured different components and each ink will have a different rate on the paper
chromatography.
What is chromatography used for?
Chromatography is most commonly used in determining and identifying what the mixture is made of
by separating the components of the mixture, these techniques are necessary for research,
separation and purification of the mixture. However, chromatography is used in a variety of different
ways such as
1. Vaccination
Chromatography helps researchers to figure out which antibodies fight against infection and disease.
Therefore, scientists used chromatography techniques to create vaccination such as the Ebola virus
to fight against infection. This is because the chromatography technique is used to determine which
antibodies are more efficient at neutralising the harmful virus.
2. Food testing
Food companies use chromatography to determine the components in their products such as
protein, vitamins and other nutrients. It might also be used to determine the nutritional quality of
the food.
3. Testing of beverage
Chromatography technique is used to verify the quality of the drink and to determine other
dangerous elements in the drink which can cause harm.
4. Drug testing
The chromatographic methods are used to accurately determine the components of the blood and
also to identify if the patient who has passed away had taken any alcohol or drugs as well as if they
had been poisoned.
5. Forensic testing
The forensic testing is used to identify and compare drug and ink samples, as well as biological
samples like saliva and blood and urine, which these tests help to analyse and identify using Gas
chromatography.
How does it work?
Chromatography can be used when the substance of a mixture has different solubility so that the
component of the mixture that is very soluble in the solvent will travel the most and the component
of the mixture with less soluble travel the least, therefore the component separates at the different
speed compared to each other.
,Chromatography has two phases: the stationary phase is the paper that does not move, and the
mobile phase is the solvent that can be liquid or gaseous. The solvent will travel up through the
chromatography paper carrying different components of the mixture on the paper chromatogram, in
which the components of the mixture are separated based on how much of
each part dissolves into the mobile phase and moves up the paper.
Chromatography works by adding the mixture to the stationary phases, while
the mobile phase helps in the movement of the mixture through the
stationary phases; when the mixture moves through the stationary phases.
The different components move at different rates because of the size and
polarity of the component. Before I start the practical, I will do a trial to make
sure if the ink chromatography works as I expected. The ink of the marker is a
mixture because it is made of several different colours. Therefore, I start by
drawing a straight line using a ruler on a piece of paper and then smear the
ink with solvent (perfume). Therefore, after some time the components of
the mixture separated into different components.
There are a variety of factors that can affect the separation of a mixture, as a result, this can affect
the Rf value. These factors that affect the separation of chromatography include:
- Adsorption
- Polarity of solvent
- Temperature
- Quality of paper chromatography
- Different paper chromatography
- Thickness of the paper chromatography
Adsorption
Adsorption is defined as the ability of a component of a mixture to attach to the stationary phases,
whereas solubility is defined as the ability of a component of a mixture to dissolve in the mobile
phase. Therefore, the higher adsorption to the stationary phases, the slower the molecules will move
through the paper, whereas the higher solubility in the mobile phases, the faster the molecules will
move through the paper chromatogram. Furthermore, if a pigment is colourless then different
methods can be used, such as shining UV light on the paper to show dots or spraying (Ninhydrin) a
component that reacts with the dots to give them colour. So, the distance travelled by each dot is
measured relative to the distance the solvent travels, which is known as the retention factor (Rf
value). The Rf is a measure of how far substance and solvent travel up the paper chromatography. It's
useful to identify a mixture because the Rf value can be compared to the known mixture and
determine its purity. Therefore, a solvent that has strong interaction with molecules in the mixture
will move further up the paper, so different solvent has different effects on paper chromatography,
and it also depends on the size of molecules and polarity of the solvent. In addition, the temperature
has an impact on component separation in all methods of chromatography. The heat transfers more
energy to the mobile phase as the temperature increases which allows the molecules in the solvent
to escape, hence the higher the temperature, the greater the solubility of the material.
The main purpose of the chromatography is to calculate the Rf value and compare it to the published
Rf value to see if the experiment is reliable and reproducible. In order to measure the Rf value, start
by drawing a 1 or 2cm pencil line at the bottom of the paper chromatography or TLC paper and place
the pigment or the mixture in the centre of the paper. Next, pour the solvent into the beaker and
gently place the paper inside the beaker making sure that the solvent level is not above the starting
line because the pigment will dissolve in the solvent. Once the pigment elutes up the paper, quickly
remove and mark the solvent front. Next measure the distance solvent travel and distance pigment
, travel up the paper and calculate the Rf value using the equation: Rf value = distance travel by
substance/distance travelled by solvent
Polarity of solvent
The size and the polarity of the molecules can affect the distance travelled by the substance. For
example, large molecules take longer to move up the paper chromatography, but small molecules are
faster up the paper. Moreover, the polarity of the molecules, as well as the type of solvent, can affect
how the spot moves. This is because different pigments and mixtures have different affinities for the
solvent and paper chromatography, meaning that it depends on how polar and nonpolar the mixture
is and how strong is the attraction between the solvent or the paper chromatography. For example,
the thin layer chromatography is made up of silica gel which is a polar compound. Therefore, the
affinity of the leaf pigment depends on the polarity of solvent, TLC paper and leaf pigment. For
example, if a more polar solvent is used it will compete better with the polar stationary phases,
meaning that the spots will partition more in favour of the mobile and will travel the faster which will
have higher Rf value. Furthermore, cellulose is used in thin layer chromatography paper because it
has shorter migration time and more concentrated substance spot, which will increase accuracy of
the experiment. Therefore, as the thin layer chromatography paper is placed inside the beaker or the
container the solvent will elute up the plate which will carry different components of mixture.
Analysing this further, the more polar mobile phases is, the faster and easier the component of the
mixture will move up the paper because TLC contain silica-coated which is more polar as it will
strongly interact with the polar components of the mixture, so if the mixture or the pigment is more
polar it will adhere to the silica and travel less distance than the component will less or nonpolar.
This is because polar molecules are attached strongly with dipole-dipoles, which means that one end
of the molecules is slightly negative and the other end is slightly positive so opposite charges attach
each other.
Effect of lid on chromatography paper
To make sure that the beaker’s atmosphere is saturated with solvent vapour, the container must be
covered by a lid. As a result, it prevents the solvent from evaporating as it moves up the paper
chromatography. Also each component of the mixture has a different rate of evaporation, due to
different boiling points and partial pressure at room temperature. Thus, an open beaker will cause
the components to separate more quickly from the mixture in chromatography. Therefore, by placing
a lid on top of the beaker it will stop evaporation and the molecules will not escape from the surface
of the solvent.
Temperature
Temperature can affect the separation of the components of the mixture because if the temperature
rises then the heat transfers more energy into the solvent which will increase the kinetic energy of
the molecules in the solvent and eventually escape from the surface of the solvent. Moreover, it can
also affect the retention time because if the temperature increases, then the retention time will
decrease as the mixture will stay in the starting line and will not separate efficiently. In addition, as
the temperature increases the density of the solvent and the elution strength of the molecules will
be reduced. This is because the heat transfers more energy to the mobile phase, and the molecules
in the solvent will evaporate and escape from the solvent.
Pros and cons of paper chromatography and TLC paper chromatography
Paper chromatography is cheaper compared to thin layer chromatography paper and easy to handle
and store, but TLC paper is sensitive and therefore, should be handled with care such as wearing
gloves to avoid contamination as the paper contains silica-coated gel. In addition, paper
chromatography is more efficient for polar substance and solvent whereas, in TLC paper the
separation will be more efficient with a less polar substance. In addition, the thin layer of
My name is Marwa and I am a newly appointed technical assistant for Chemicalequip, in this
assignment, I will carry out different chromatographic techniques such as ink paper chromatography,
chromatography of leaf pigments, thin-layer chromatography and paper chromatography of amino
acid. Therefore, I will demonstrate my skill to be able to perform a variety of practical procedures
and techniques for separating and identifying components in a mixture. I will also describe the
method, and types of equipment, analyse and evaluate the results, compare my results with
published results and suggest improvements.
What is chromatography?
Mikhail Tswett discovered chromatography during his studies on plant pigment in 1901. He applied
the method and the technique on how to separate plant pigment including chlorophyll, xanthophyll,
and carotenoid. Chromatography is a laboratory technique that is used to separate a mixture of
different components such as ink chromatography which is used to analyse the mixture by separating
the ink into what they are made of, which means that each component of the mixture will move at
different rates as the paper is placed inside the beaker. This will cause the mixture to separate out
into coloured different components and each ink will have a different rate on the paper
chromatography.
What is chromatography used for?
Chromatography is most commonly used in determining and identifying what the mixture is made of
by separating the components of the mixture, these techniques are necessary for research,
separation and purification of the mixture. However, chromatography is used in a variety of different
ways such as
1. Vaccination
Chromatography helps researchers to figure out which antibodies fight against infection and disease.
Therefore, scientists used chromatography techniques to create vaccination such as the Ebola virus
to fight against infection. This is because the chromatography technique is used to determine which
antibodies are more efficient at neutralising the harmful virus.
2. Food testing
Food companies use chromatography to determine the components in their products such as
protein, vitamins and other nutrients. It might also be used to determine the nutritional quality of
the food.
3. Testing of beverage
Chromatography technique is used to verify the quality of the drink and to determine other
dangerous elements in the drink which can cause harm.
4. Drug testing
The chromatographic methods are used to accurately determine the components of the blood and
also to identify if the patient who has passed away had taken any alcohol or drugs as well as if they
had been poisoned.
5. Forensic testing
The forensic testing is used to identify and compare drug and ink samples, as well as biological
samples like saliva and blood and urine, which these tests help to analyse and identify using Gas
chromatography.
How does it work?
Chromatography can be used when the substance of a mixture has different solubility so that the
component of the mixture that is very soluble in the solvent will travel the most and the component
of the mixture with less soluble travel the least, therefore the component separates at the different
speed compared to each other.
,Chromatography has two phases: the stationary phase is the paper that does not move, and the
mobile phase is the solvent that can be liquid or gaseous. The solvent will travel up through the
chromatography paper carrying different components of the mixture on the paper chromatogram, in
which the components of the mixture are separated based on how much of
each part dissolves into the mobile phase and moves up the paper.
Chromatography works by adding the mixture to the stationary phases, while
the mobile phase helps in the movement of the mixture through the
stationary phases; when the mixture moves through the stationary phases.
The different components move at different rates because of the size and
polarity of the component. Before I start the practical, I will do a trial to make
sure if the ink chromatography works as I expected. The ink of the marker is a
mixture because it is made of several different colours. Therefore, I start by
drawing a straight line using a ruler on a piece of paper and then smear the
ink with solvent (perfume). Therefore, after some time the components of
the mixture separated into different components.
There are a variety of factors that can affect the separation of a mixture, as a result, this can affect
the Rf value. These factors that affect the separation of chromatography include:
- Adsorption
- Polarity of solvent
- Temperature
- Quality of paper chromatography
- Different paper chromatography
- Thickness of the paper chromatography
Adsorption
Adsorption is defined as the ability of a component of a mixture to attach to the stationary phases,
whereas solubility is defined as the ability of a component of a mixture to dissolve in the mobile
phase. Therefore, the higher adsorption to the stationary phases, the slower the molecules will move
through the paper, whereas the higher solubility in the mobile phases, the faster the molecules will
move through the paper chromatogram. Furthermore, if a pigment is colourless then different
methods can be used, such as shining UV light on the paper to show dots or spraying (Ninhydrin) a
component that reacts with the dots to give them colour. So, the distance travelled by each dot is
measured relative to the distance the solvent travels, which is known as the retention factor (Rf
value). The Rf is a measure of how far substance and solvent travel up the paper chromatography. It's
useful to identify a mixture because the Rf value can be compared to the known mixture and
determine its purity. Therefore, a solvent that has strong interaction with molecules in the mixture
will move further up the paper, so different solvent has different effects on paper chromatography,
and it also depends on the size of molecules and polarity of the solvent. In addition, the temperature
has an impact on component separation in all methods of chromatography. The heat transfers more
energy to the mobile phase as the temperature increases which allows the molecules in the solvent
to escape, hence the higher the temperature, the greater the solubility of the material.
The main purpose of the chromatography is to calculate the Rf value and compare it to the published
Rf value to see if the experiment is reliable and reproducible. In order to measure the Rf value, start
by drawing a 1 or 2cm pencil line at the bottom of the paper chromatography or TLC paper and place
the pigment or the mixture in the centre of the paper. Next, pour the solvent into the beaker and
gently place the paper inside the beaker making sure that the solvent level is not above the starting
line because the pigment will dissolve in the solvent. Once the pigment elutes up the paper, quickly
remove and mark the solvent front. Next measure the distance solvent travel and distance pigment
, travel up the paper and calculate the Rf value using the equation: Rf value = distance travel by
substance/distance travelled by solvent
Polarity of solvent
The size and the polarity of the molecules can affect the distance travelled by the substance. For
example, large molecules take longer to move up the paper chromatography, but small molecules are
faster up the paper. Moreover, the polarity of the molecules, as well as the type of solvent, can affect
how the spot moves. This is because different pigments and mixtures have different affinities for the
solvent and paper chromatography, meaning that it depends on how polar and nonpolar the mixture
is and how strong is the attraction between the solvent or the paper chromatography. For example,
the thin layer chromatography is made up of silica gel which is a polar compound. Therefore, the
affinity of the leaf pigment depends on the polarity of solvent, TLC paper and leaf pigment. For
example, if a more polar solvent is used it will compete better with the polar stationary phases,
meaning that the spots will partition more in favour of the mobile and will travel the faster which will
have higher Rf value. Furthermore, cellulose is used in thin layer chromatography paper because it
has shorter migration time and more concentrated substance spot, which will increase accuracy of
the experiment. Therefore, as the thin layer chromatography paper is placed inside the beaker or the
container the solvent will elute up the plate which will carry different components of mixture.
Analysing this further, the more polar mobile phases is, the faster and easier the component of the
mixture will move up the paper because TLC contain silica-coated which is more polar as it will
strongly interact with the polar components of the mixture, so if the mixture or the pigment is more
polar it will adhere to the silica and travel less distance than the component will less or nonpolar.
This is because polar molecules are attached strongly with dipole-dipoles, which means that one end
of the molecules is slightly negative and the other end is slightly positive so opposite charges attach
each other.
Effect of lid on chromatography paper
To make sure that the beaker’s atmosphere is saturated with solvent vapour, the container must be
covered by a lid. As a result, it prevents the solvent from evaporating as it moves up the paper
chromatography. Also each component of the mixture has a different rate of evaporation, due to
different boiling points and partial pressure at room temperature. Thus, an open beaker will cause
the components to separate more quickly from the mixture in chromatography. Therefore, by placing
a lid on top of the beaker it will stop evaporation and the molecules will not escape from the surface
of the solvent.
Temperature
Temperature can affect the separation of the components of the mixture because if the temperature
rises then the heat transfers more energy into the solvent which will increase the kinetic energy of
the molecules in the solvent and eventually escape from the surface of the solvent. Moreover, it can
also affect the retention time because if the temperature increases, then the retention time will
decrease as the mixture will stay in the starting line and will not separate efficiently. In addition, as
the temperature increases the density of the solvent and the elution strength of the molecules will
be reduced. This is because the heat transfers more energy to the mobile phase, and the molecules
in the solvent will evaporate and escape from the solvent.
Pros and cons of paper chromatography and TLC paper chromatography
Paper chromatography is cheaper compared to thin layer chromatography paper and easy to handle
and store, but TLC paper is sensitive and therefore, should be handled with care such as wearing
gloves to avoid contamination as the paper contains silica-coated gel. In addition, paper
chromatography is more efficient for polar substance and solvent whereas, in TLC paper the
separation will be more efficient with a less polar substance. In addition, the thin layer of
