SOMERSBY HOLMES
BTEC L3 EXTENDED DIPLOMA IN APPLIED SCIENCE
Unit 2: Practical Scientific Procedures and
Techniques
Assignment 2C: Separate to identify
Introduction
What is chromatography?
Chromatography is an analytical technique that enables us to separate
substances in mixtures, especially complex mixtures.
How does chromatography work?
The substances are separated in mixtures by dissolving them in mobile phases
(where the molecules can move), e.g. solvent, this allows them to move on to
the stationary phase (where the molecules cannot move), e.g. paper. They
separate according to their relative affinity – how strongly attracted they are to
other molecules. Their affinity depends on 2 properties: adsorption and solubility.
The higher the substance’s adsorption to the stationary phase, the slower it will
travel through the column, whereas, the higher the substance’s solubility to the
mobile phase, the faster it will travel through the column.
The distance that the substances move highly depend on its solubility in the
mobile phase and its retention by the stationary phase. The differences in
solubility and retention allow these substances to separate. The higher a
substance’s solubility, the further it will travel and therefore the higher its R f
value. These substances will travel at different speeds depending on many
factors including the solubility of the substance in the mobile phase.
Why is chromatography important?
The importance of chromatography is that it allows chemists to separate, identify
and purify substances in a mixture. This is important as it can further be used for
chemical analysis in industry.
What factors affect the separation of components in a mixture?
- Affinity, e.g. if a substance has a high affinity then it will move faster
through the column, resulting in a higher R f value, however if it has a low
affinity then it will take longer for it to move through the column, meaning
a low Rf value
- Polarity, e.g. more polar molecules will have a stronger attraction to polar
solvents, therefore will travel further as opposed to a non-polar solvent
- Solubility, e.g. if a substance is very insoluble in the mobile phase, it will
be hard for it to pass through to the stationary phase, however if the
solubility of a substance is high in the mobile phase, it will be able to move
faster to the stationary phase, resulting in a high R f value and a faster rate
of separation
- Surface area of stationary phase, e.g. having a large surface area will
allow the substances to separate out further, enabling them to progress
past the stationary phase
1
, SOMERSBY HOLMES
BTEC L3 EXTENDED DIPLOMA IN APPLIED SCIENCE
Unit 2: Practical Scientific Procedures and
Techniques
- Temperature, e.g. having a change in temperature, will affect how
fast/slow the substances travel up the chromatogram which will further
affect their Rf value
- Type of stationary phase, e.g. the running solvent will affect the speed at
which substances are separated and how soluble the substances are in the
stationary phase
- Molecule size, e.g. larger molecules will take longer to travel as opposed
to smaller molecules which are more mobile
Types of chromatography and how they work:
Size exclusion chromatography is a type of chromatography technique which
works by separating molecules in terms of size through porous beads - molecules
that are larger in size are separated first and the molecules that are smaller in
size are separated last.
Affinity chromatography is another type of chromatography technique which
separates specific molecules or a group of molecules from complex mixtures. It
works in terms of biological interactions between molecules, for example
between an enzyme and a substrate. In this example, the enzyme and the
substrate will bind, allowing the non-bound molecules/substances to pass
through the solution.
Ion exchange chromatography is a technique used in chromatography which
separates charged molecules, e.g. proteins, nucleotides. It works by passing the
sample of charged molecules through a column where molecules will bind to
oppositely charged sites in the stationary phase.
Gas chromatography is another technique used in chromatography which
separates volatile mixtures using an unreactive gas like helium. The time the
molecules get separated are recorded – this is known as the retention time. They
get detected once they are separated and appear as a peak on the GLC trace.
Partition chromatography is a chromatography technique used to separate
mixtures into 2 liquid phases. It works by shifting the mobile phase to the
stationary phase. When this happens, the substances are separated through
capillary action.
Paper chromatography
Paper chromatography is the most basic chromatography technique which is
used to separate substances in a mixture. It works by dissolving the substances
in the mobile phase, which is usually an aqueous (water-based) solvent or non-
aqueous organic (carbon-based) solvent (e.g. propanone), and passing them
through to the stationary phase, which is usually absorbent paper. For each
substance in the mixture, there is dynamic equilibrium between the stationary
and mobile phase. The chromatogram produced will show the separation of the
substances and this separation occurs as a result of the substance’s relative
affinity to the mobile and stationary phase. The solvent will move up the paper
by capillary action.
2
BTEC L3 EXTENDED DIPLOMA IN APPLIED SCIENCE
Unit 2: Practical Scientific Procedures and
Techniques
Assignment 2C: Separate to identify
Introduction
What is chromatography?
Chromatography is an analytical technique that enables us to separate
substances in mixtures, especially complex mixtures.
How does chromatography work?
The substances are separated in mixtures by dissolving them in mobile phases
(where the molecules can move), e.g. solvent, this allows them to move on to
the stationary phase (where the molecules cannot move), e.g. paper. They
separate according to their relative affinity – how strongly attracted they are to
other molecules. Their affinity depends on 2 properties: adsorption and solubility.
The higher the substance’s adsorption to the stationary phase, the slower it will
travel through the column, whereas, the higher the substance’s solubility to the
mobile phase, the faster it will travel through the column.
The distance that the substances move highly depend on its solubility in the
mobile phase and its retention by the stationary phase. The differences in
solubility and retention allow these substances to separate. The higher a
substance’s solubility, the further it will travel and therefore the higher its R f
value. These substances will travel at different speeds depending on many
factors including the solubility of the substance in the mobile phase.
Why is chromatography important?
The importance of chromatography is that it allows chemists to separate, identify
and purify substances in a mixture. This is important as it can further be used for
chemical analysis in industry.
What factors affect the separation of components in a mixture?
- Affinity, e.g. if a substance has a high affinity then it will move faster
through the column, resulting in a higher R f value, however if it has a low
affinity then it will take longer for it to move through the column, meaning
a low Rf value
- Polarity, e.g. more polar molecules will have a stronger attraction to polar
solvents, therefore will travel further as opposed to a non-polar solvent
- Solubility, e.g. if a substance is very insoluble in the mobile phase, it will
be hard for it to pass through to the stationary phase, however if the
solubility of a substance is high in the mobile phase, it will be able to move
faster to the stationary phase, resulting in a high R f value and a faster rate
of separation
- Surface area of stationary phase, e.g. having a large surface area will
allow the substances to separate out further, enabling them to progress
past the stationary phase
1
, SOMERSBY HOLMES
BTEC L3 EXTENDED DIPLOMA IN APPLIED SCIENCE
Unit 2: Practical Scientific Procedures and
Techniques
- Temperature, e.g. having a change in temperature, will affect how
fast/slow the substances travel up the chromatogram which will further
affect their Rf value
- Type of stationary phase, e.g. the running solvent will affect the speed at
which substances are separated and how soluble the substances are in the
stationary phase
- Molecule size, e.g. larger molecules will take longer to travel as opposed
to smaller molecules which are more mobile
Types of chromatography and how they work:
Size exclusion chromatography is a type of chromatography technique which
works by separating molecules in terms of size through porous beads - molecules
that are larger in size are separated first and the molecules that are smaller in
size are separated last.
Affinity chromatography is another type of chromatography technique which
separates specific molecules or a group of molecules from complex mixtures. It
works in terms of biological interactions between molecules, for example
between an enzyme and a substrate. In this example, the enzyme and the
substrate will bind, allowing the non-bound molecules/substances to pass
through the solution.
Ion exchange chromatography is a technique used in chromatography which
separates charged molecules, e.g. proteins, nucleotides. It works by passing the
sample of charged molecules through a column where molecules will bind to
oppositely charged sites in the stationary phase.
Gas chromatography is another technique used in chromatography which
separates volatile mixtures using an unreactive gas like helium. The time the
molecules get separated are recorded – this is known as the retention time. They
get detected once they are separated and appear as a peak on the GLC trace.
Partition chromatography is a chromatography technique used to separate
mixtures into 2 liquid phases. It works by shifting the mobile phase to the
stationary phase. When this happens, the substances are separated through
capillary action.
Paper chromatography
Paper chromatography is the most basic chromatography technique which is
used to separate substances in a mixture. It works by dissolving the substances
in the mobile phase, which is usually an aqueous (water-based) solvent or non-
aqueous organic (carbon-based) solvent (e.g. propanone), and passing them
through to the stationary phase, which is usually absorbent paper. For each
substance in the mixture, there is dynamic equilibrium between the stationary
and mobile phase. The chromatogram produced will show the separation of the
substances and this separation occurs as a result of the substance’s relative
affinity to the mobile and stationary phase. The solvent will move up the paper
by capillary action.
2
