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, Chromatography
Chromatography is used to separate individual components from a mixture of
substances. All forms of chromatography have a stationary & mobile phase.
The stationary phase does not move & is normally a solid or a liquid
supported on a solid.
The mobile phase does move & is normally a liquid or a gas
Substances that can be separated by chromatography have the property of
adsorption which allows the separation.
Thin layer chromatography (TLC)
TLC is a quick & inexpensive analytical technique that indicates how many
components are in a mixture. The technique uses a TLC plate which is usually
a plastic sheet or glass, coated with a thin layer of a solid adsorbent
substance- usually silica.
In TLC, the adsorbent is the stationary phase. The different components in
the mixture have different affinities for the adsorbent & bind with
differing strengths to its surface. Adsorption is the process by which the
solid silica holds the different substances in the mixture to its surface.
Thin layer chromatograms are analysed by calculating the retention factor,
R f, & comparing it to known R f values using the same solvent system &
adsorbent. 𝒅𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒎𝒐𝒗𝒆𝒅 𝒃𝒚 𝒕𝒉𝒆 𝒄𝒐𝒎𝒑𝒐𝒏𝒆𝒏𝒕
Rf = 𝒅𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒎𝒐𝒗𝒆𝒅 𝒃𝒚 𝒕𝒉𝒆 𝒔𝒐𝒍𝒗𝒆𝒏𝒕 𝒇𝒓𝒐𝒏𝒕
, Gas chromatography
Gas chromatography is useful for separating & identifying volatile organic
compounds present in a mixture.
The stationary phase is a high B.P liquid adsorbed onto an inert solid
support. The mobile phase is an inert carrier gas e.g. helium or neon.
1.A small amount of the volatile mixture
is injected into the gas chromatogram
2.The mobile carrier gas carries the
components in the sample through the
capillary column which contains the
liquid stationary phase absorbed onto
the solid support
3.The components slow down as they
interact with the liquid stationary phase
inside the column. The more soluble the
component is in the liquid stationary
Column oven phase, the slower it moves through the
Gas chromatogram capillary column.
4.The components of the mixture are separated depending on their solubility in the
liquid stationary phase which causes the different compounds to reach the
detector at different times.
5.The compound retained in the column for the shortest time has the lowest
retention time & is detected 1 st. The retention time is the time taken for each
component to travel through the column.
, Gas chromatogram
Each component is detected as a peak on a gas chromatogram. 2 pieces of
information can be obtained from a gas chromatogram:
• Retention times can be used to identify the components present in the
sample by comparing these to retention times for known components.
• Peak integrations, which are the areas under each peak, can be used to
determine the concentrations of components in the sample by comparing
the values with values obtained from standard solutions of the
components. Procedure:
1. Prepare standard solution of known concentrations of
the compound being investigated
2.Obtain gas chromatogram for each standard solution
3.Plot a calibration curve of peak area against
concentration. This is called external calibration &
offers a method for converting peak area into
concentration.
4.Obtain a gas chromatogram of the compound being
investigated under the same conditions.
5.Use the calibration curve to measure the
concentrations of the compound
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