2.3 Chromatography
Investigation Using Thin Layer Chromatography, Paper Chromatography and Amino acids
to separate photosynthetic pigments
,Introduction
Chromatography
Chromatography is a method for separating components in a mixture (or solutes) by how
they distribute between two phases. One phase known as the mobile phase moves and
can be a liquid or gas. The other known as the stationary phase remains fixed and can be a
solid or liquid. Each component’s movements between these phases depends on its
properties.
Theory of Chromatography
explain what it is and talk about in my justification for each why it should or not be used
instead.
Column chromatography (CC):
• A column is used with a stationary phase (e.g silica gel or paper) and a solvent as
the mobile phase.
• Very useful for extracting larger quantities of pigments.
• The pigments are separated based on their affinities for the mobile and stationary
phase.
• The experiment is quick should take around 15 -20minutes
• Relatively cheap
High- performance liquid chromatography (HPLC):
• Very expensive
• Advanced technique that provides precision and quantitative research
• High pressure is used to push the solvent through a column packed with fine
particles
• Pigments are easily separated based on their molecular size, affinities (to the
stationary phase and its interactions with the solvent (mobile phase)) and
polarity
• Accurate photosynthetic pigments were achieved for the experiment.
• Relatively rapid
, Gas chromatography (GC):
• Used to separate pigments that are volatile or can be made volatile through a
chemical process called derivatization
• In gas chromatography the pigments are vaporized and carried by inert gases
eg. Nitrogen and helium)
• Based on volatility and interaction of each pigment with the stationary phase
and mobile phase separations occurs.
• Relatively cheap
• Fairly quick
Ion-Exchange Chromatography (IEC):
• Pigments are separated based on charges in this technique. This is done by
exploiting the interaction between charged molecules and oppositely charged
stationary phase
• This technique is not commonly used for photosynthetic pigments/compounds
however it has the ability to isolate specific charged molecules.
• This technique is moderately expensive
• This technique can take hours to complete>
Aim of the experiment:
The aim is to extract photosynthetic pigments from the spinach leaves used in the
experiments and separate them using thin layer chromatography. This method allows me
to be able to see and depict the various pigments extracted from a single extract. This also
allows identification through rf value calculation and color observation on the different
pigments.
This experiment demonstrates practical skills in chromatography focusing on the
separation and identification of compounds from a biological sample.
, How Does TLC Chromatography Work?
Compounds in a mixture can be separated and identified using thin-layer chromatography.
The pigments begin to develop and separate (after the mixture was added to TLC’s base
line). The plate is placed in a glass jar after with a solvent (propanone) flowing vertically up
the plate. Strong interactions between compounds (like silica) that have polarities like the
stationary phase will cause them to move more slowly and have lower Rf values. On the
other hand, because of their weaker interactions with the polar stationary phase, less polar
molecules have larger Rf values and travel further up the plate. Once the solvent front
reaches the preferred height the plate is removed and the separated spots/pigments
representing different compounds can be calculated by the retention factor.
Real Life Applications Of TLC
1. Purity Testing: By identifying impurities TLC can verify the purity of compounds.
2. Compound Identification: The retention factor is used to help identify compounds.
3. Quality Control: TLC is used by pharmaceutical businesses to verify purity of
medicinal products as a measure of quality control.
Strengths and Limitations of using TLC Chromatography
Strengths:
1. Simplicity and Speed- TLC is a simple and rapid method that needs very little
equipment. Findings are usually available in a few hours.
2. Cost-Effective- When compared to other chromatographic methods like HPLC or
GC the equipment's required for the TLC is reasonably inexpensive. Due to this
reasoning, Schools have access to TLC and can conduct the experiments more
frequently.
Limitations:
1. Limited Quantitative Data: TLC is mostly a qualitative method which means that
it can show that chemicals are present it cannot give exact numerical data.
2. Low Resolution: When compared to other chromatographic techniques like
HPLC or GC, TLC has a lower resolution. This could lead to patches in the
chromatogram's result making it more difficult to see the different spots/
pigments.
Investigation Using Thin Layer Chromatography, Paper Chromatography and Amino acids
to separate photosynthetic pigments
,Introduction
Chromatography
Chromatography is a method for separating components in a mixture (or solutes) by how
they distribute between two phases. One phase known as the mobile phase moves and
can be a liquid or gas. The other known as the stationary phase remains fixed and can be a
solid or liquid. Each component’s movements between these phases depends on its
properties.
Theory of Chromatography
explain what it is and talk about in my justification for each why it should or not be used
instead.
Column chromatography (CC):
• A column is used with a stationary phase (e.g silica gel or paper) and a solvent as
the mobile phase.
• Very useful for extracting larger quantities of pigments.
• The pigments are separated based on their affinities for the mobile and stationary
phase.
• The experiment is quick should take around 15 -20minutes
• Relatively cheap
High- performance liquid chromatography (HPLC):
• Very expensive
• Advanced technique that provides precision and quantitative research
• High pressure is used to push the solvent through a column packed with fine
particles
• Pigments are easily separated based on their molecular size, affinities (to the
stationary phase and its interactions with the solvent (mobile phase)) and
polarity
• Accurate photosynthetic pigments were achieved for the experiment.
• Relatively rapid
, Gas chromatography (GC):
• Used to separate pigments that are volatile or can be made volatile through a
chemical process called derivatization
• In gas chromatography the pigments are vaporized and carried by inert gases
eg. Nitrogen and helium)
• Based on volatility and interaction of each pigment with the stationary phase
and mobile phase separations occurs.
• Relatively cheap
• Fairly quick
Ion-Exchange Chromatography (IEC):
• Pigments are separated based on charges in this technique. This is done by
exploiting the interaction between charged molecules and oppositely charged
stationary phase
• This technique is not commonly used for photosynthetic pigments/compounds
however it has the ability to isolate specific charged molecules.
• This technique is moderately expensive
• This technique can take hours to complete>
Aim of the experiment:
The aim is to extract photosynthetic pigments from the spinach leaves used in the
experiments and separate them using thin layer chromatography. This method allows me
to be able to see and depict the various pigments extracted from a single extract. This also
allows identification through rf value calculation and color observation on the different
pigments.
This experiment demonstrates practical skills in chromatography focusing on the
separation and identification of compounds from a biological sample.
, How Does TLC Chromatography Work?
Compounds in a mixture can be separated and identified using thin-layer chromatography.
The pigments begin to develop and separate (after the mixture was added to TLC’s base
line). The plate is placed in a glass jar after with a solvent (propanone) flowing vertically up
the plate. Strong interactions between compounds (like silica) that have polarities like the
stationary phase will cause them to move more slowly and have lower Rf values. On the
other hand, because of their weaker interactions with the polar stationary phase, less polar
molecules have larger Rf values and travel further up the plate. Once the solvent front
reaches the preferred height the plate is removed and the separated spots/pigments
representing different compounds can be calculated by the retention factor.
Real Life Applications Of TLC
1. Purity Testing: By identifying impurities TLC can verify the purity of compounds.
2. Compound Identification: The retention factor is used to help identify compounds.
3. Quality Control: TLC is used by pharmaceutical businesses to verify purity of
medicinal products as a measure of quality control.
Strengths and Limitations of using TLC Chromatography
Strengths:
1. Simplicity and Speed- TLC is a simple and rapid method that needs very little
equipment. Findings are usually available in a few hours.
2. Cost-Effective- When compared to other chromatographic methods like HPLC or
GC the equipment's required for the TLC is reasonably inexpensive. Due to this
reasoning, Schools have access to TLC and can conduct the experiments more
frequently.
Limitations:
1. Limited Quantitative Data: TLC is mostly a qualitative method which means that
it can show that chemicals are present it cannot give exact numerical data.
2. Low Resolution: When compared to other chromatographic techniques like
HPLC or GC, TLC has a lower resolution. This could lead to patches in the
chromatogram's result making it more difficult to see the different spots/
pigments.
