Assignment 2C - Chromatography of Amino acids.
Introduction
Chromatography is a technique used for separating components or solutes of a mixture based on the relative amounts of each
solute distributed between a moving fluid stream, called the mobile phase, and a contiguous stationary phase (1). The
separation process relies on intermolecular forces such as Van der Waals forces, hydrogen bonding, and dipole-dipole
interactions, which influence how long a molecule stays in the stationary phase. Larger molecules may move more slowly due
to steric hindrance, while elongated molecules might interact more extensively with the stationary phase. Factors such as pH of
the solvent, solvent polarity, temperature, and flow rate can impact the efficiency and resolution of chromatography. Polarity, in
particular, plays a crucial role, affecting both the solubility of molecules in the mobile phase and their retention in the stationary
phase. Components with stronger interactions with the stationary phase move slower, while those with stronger interactions
with the mobile phase move faster, leading to separation. Developed by Russian botanist Mikhail Tsvet in the early 20th century
for separating plant pigments, chromatography has evolved into various methods, including gas chromatography (GC),
high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC), and is now integral in fields like
biochemistry, analytical chemistry, and pharmaceuticals. For this task, we performed two types of chromatography: paper
chromatography and thin-layer chromatography (TLC). In TLC, a thin layer of substance on a flat surface is placed in a liquid,
which travels up the surface, carrying the substances with it and allowing them to separate based on their different interactions
with the stationary phase. In paper chromatography, a solvent moves up a piece of paper via capillary action, carrying the
dissolved substances, and separating them based on their migration distances. In both methods, the Rf value is calculated by
measuring the points where each component of the compound stops moving along the TLC plate or the paper.
Chromatography's ability to separate and analyse complex mixtures with high precision makes it a fundamental technique in
modern science.
In our chromatography experiments, we used both paper chromatography and thin-layer chromatography (TLC) to separate
mixtures based on how components interact with a mobile phase (fluid) and a stationary phase. By choosing hexane
(non-polar) and acetone (polar) in a specific ratio, we optimised separation for various compounds: hexane for non-polar and
acetone for polar compounds.
To ensure accuracy, we conducted control experiments using known standards, confirming our method's reliability. For
example, matching the Rf value for leucine with expected values validated our approach. TLC involves applying substances
onto a flat surface, while paper chromatography uses paper as the substrate. Both methods separate substances based on
how far they travel with the solvent, and the Rf value indicates each component's position. Chromatography is crucial for
precise analysis of complex mixtures.
The calculation is:
Rf = distance moved by the component / distance moved by the solvent.
How to carry out simple paper chromatography:
Compounds can be separated using paper chromatography according to their varying solubility in the stationary phase and
mobile phase (solvent). It works very well for compounds with colour but also can work very well for substances that don't have
any colour, an example of this is amino acids we could use a fume box or indicators to make them clear to us. The two distinct
stages: the mobile phase and the stationary phase. The solvent that passes through the paper (stationary phase) and conveys
the mixture's constituent parts is known as the mobile phase; the solid substance that absorbs the mixture as it passes through
is known as the mobile phase. Due to the mixture's constituents' varying degrees of attraction to the two phases, the various
substances flow through the paper at different speeds.
Factors that affect Paper Chromatography:
The type of solvent used This is because the solubility of each compound is varied and is very different.
Quality of the paper used Some papers could have better absorption and retention which could affect the
separation.
The concentration of the sample How diluted or how strong affects how much we can see it on the paper and
whether it would be difficult for us too see e.g if the liquid was too dilute it would be
hard for us to see the separation happening properly at each of the stops.
Temperature Each substance has their own individual ways of reacting and temperature can
affect all of them differently which in the end can even affect the separation.
Flow Rate The speed at which the mobile phase moves through the stationary phase can
impact the separation. A slower flow rate typically allows for better separation as
analytes have more time to interact with the stationary phase.
Introduction
Chromatography is a technique used for separating components or solutes of a mixture based on the relative amounts of each
solute distributed between a moving fluid stream, called the mobile phase, and a contiguous stationary phase (1). The
separation process relies on intermolecular forces such as Van der Waals forces, hydrogen bonding, and dipole-dipole
interactions, which influence how long a molecule stays in the stationary phase. Larger molecules may move more slowly due
to steric hindrance, while elongated molecules might interact more extensively with the stationary phase. Factors such as pH of
the solvent, solvent polarity, temperature, and flow rate can impact the efficiency and resolution of chromatography. Polarity, in
particular, plays a crucial role, affecting both the solubility of molecules in the mobile phase and their retention in the stationary
phase. Components with stronger interactions with the stationary phase move slower, while those with stronger interactions
with the mobile phase move faster, leading to separation. Developed by Russian botanist Mikhail Tsvet in the early 20th century
for separating plant pigments, chromatography has evolved into various methods, including gas chromatography (GC),
high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC), and is now integral in fields like
biochemistry, analytical chemistry, and pharmaceuticals. For this task, we performed two types of chromatography: paper
chromatography and thin-layer chromatography (TLC). In TLC, a thin layer of substance on a flat surface is placed in a liquid,
which travels up the surface, carrying the substances with it and allowing them to separate based on their different interactions
with the stationary phase. In paper chromatography, a solvent moves up a piece of paper via capillary action, carrying the
dissolved substances, and separating them based on their migration distances. In both methods, the Rf value is calculated by
measuring the points where each component of the compound stops moving along the TLC plate or the paper.
Chromatography's ability to separate and analyse complex mixtures with high precision makes it a fundamental technique in
modern science.
In our chromatography experiments, we used both paper chromatography and thin-layer chromatography (TLC) to separate
mixtures based on how components interact with a mobile phase (fluid) and a stationary phase. By choosing hexane
(non-polar) and acetone (polar) in a specific ratio, we optimised separation for various compounds: hexane for non-polar and
acetone for polar compounds.
To ensure accuracy, we conducted control experiments using known standards, confirming our method's reliability. For
example, matching the Rf value for leucine with expected values validated our approach. TLC involves applying substances
onto a flat surface, while paper chromatography uses paper as the substrate. Both methods separate substances based on
how far they travel with the solvent, and the Rf value indicates each component's position. Chromatography is crucial for
precise analysis of complex mixtures.
The calculation is:
Rf = distance moved by the component / distance moved by the solvent.
How to carry out simple paper chromatography:
Compounds can be separated using paper chromatography according to their varying solubility in the stationary phase and
mobile phase (solvent). It works very well for compounds with colour but also can work very well for substances that don't have
any colour, an example of this is amino acids we could use a fume box or indicators to make them clear to us. The two distinct
stages: the mobile phase and the stationary phase. The solvent that passes through the paper (stationary phase) and conveys
the mixture's constituent parts is known as the mobile phase; the solid substance that absorbs the mixture as it passes through
is known as the mobile phase. Due to the mixture's constituents' varying degrees of attraction to the two phases, the various
substances flow through the paper at different speeds.
Factors that affect Paper Chromatography:
The type of solvent used This is because the solubility of each compound is varied and is very different.
Quality of the paper used Some papers could have better absorption and retention which could affect the
separation.
The concentration of the sample How diluted or how strong affects how much we can see it on the paper and
whether it would be difficult for us too see e.g if the liquid was too dilute it would be
hard for us to see the separation happening properly at each of the stops.
Temperature Each substance has their own individual ways of reacting and temperature can
affect all of them differently which in the end can even affect the separation.
Flow Rate The speed at which the mobile phase moves through the stationary phase can
impact the separation. A slower flow rate typically allows for better separation as
analytes have more time to interact with the stationary phase.
