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Unit 2 Mod 2 Chromatographic Methods of Separation

Chromatographic methods separate components of a mixture through differential partitioning between a stationary and mobile phase. There are several types including paper, thin layer, column, and gas-liquid chromatography. Paper chromatography involves placing a sample spot on chromatography paper and developing it in a mobile phase solvent, allowing components to separate based on differences in adsorption and solubility. Retention factors can identify components by the distance traveled in the mobile phase. Thin layer chromatography uses a similar process but with a dried slurry stationary phase coated on a glass plate, allowing for routine good separation. Column chromatography packs the stationary phase into a column, separating components by the time taken to reach the bottom in a mobile phase added to the top. Gas-liquid chromatography

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176 views

Unit 2 Mod 2 Chromatographic Methods of Separation

Chromatographic methods separate components of a mixture through differential partitioning between a stationary and mobile phase. There are several types including paper, thin layer, column, and gas-liquid chromatography. Paper chromatography involves placing a sample spot on chromatography paper and developing it in a mobile phase solvent, allowing components to separate based on differences in adsorption and solubility. Retention factors can identify components by the distance traveled in the mobile phase. Thin layer chromatography uses a similar process but with a dried slurry stationary phase coated on a glass plate, allowing for routine good separation. Column chromatography packs the stationary phase into a column, separating components by the time taken to reach the bottom in a mobile phase added to the top. Gas-liquid chromatography

Uploaded by

Loveena Steadman
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 6

Chromatographic methods of separation

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Theoretical principles of chromatography


This is based on the partition of components of a mixture between a
stationary phase and a mobile phase. The different components experience
slightly different adsorption forces with the stationary phase and different
solubilities in the mobile phase. Differences in both phases result in
separation of the components in the mixture.
There are several types of chromatographic methods:- paper, thin layer,
column and gas-liquid.
Chromatographic
method
Paper
Thin layer

Column

Gas-liquid

Stationary phase

Mobile phase

cellulose
Dried slurry of powdered
alumina or silica on a glass
plate
Dried slurry of powdered
alumina or silica packed
into a column
Inert powder coated with an
involatile oil

Any solvent
Any solvent

Any solvent

Nitrogen gas

Paper chromatography
The mixture is placed as a spot roughly 1 inch from the edge of the
chromatography paper. The paper is then dipped into a suitable solvent such
as water or ethanol. The solvent travels up (if it is ascending
chromatography) and via adsorption forces and solubility, the components
separate. The retention factor (Rf) can be calculated for each component.
Rf = distance traveled by component
distance traveled by solvent front
The solvent front is the distance travelled by the solvent up(or down) the
paper. All components have their own retention factor values, these values
can therefore be used to identify substances. In order for this to be useful,
the procedure must be standardized i.e. carried out inside a glass tank with a
lid to exclude draughts and the solvent would need to be buffered.

Chromatographic methods of separation

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A variation of the process is called two-way paper chromatography. After


one solvent is used, the paper is placed with the left side to the bottom and a
second solvent is used for chromatogram development. The spots are more
widely dispersed and can be recognised from their position on the final
chromatogram.

This method is used to determine the identity of amino acids, however they
must be first sprayed with a visualizing agent called ninhydrin or iodine
vapour to form coloured spots.

Chromatographic methods of separation

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Thin Layer Chromatography (TLC)


Similar to paper chromatography, however the stationary phase is on a glass
plate and is a dried slurry of powdered alumina or silica. Good separation
can be routinely achieved on these glass plates and this method can be used
to investigate the several products formed from the nitration of phenol.

Column chromatography
Similar to TLC but the stationary phase is packed into a column.

In carrying out a separation, fresh solvent is added at the


top of the column from a separating funnel dripping at the
same rate as the solvent leaves the column at the bottom,
this is to avoid air bubbles. Different components can
then be identified from the time taken for each to reach
the bottom of the column. The green mixture separates
into blue and yellow bands

Chromatographic methods of separation

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Amino acids, peptides,
nitrogen bases in nucleotides
can be identified using this
method.
One major use with this
method is the length of time
it takes for separation, since
this is done under gravity
and the particle size in the
stationary phase is very
small, separation takes an
unacceptably long time using
industry standards.

Gas-Liquid Chromatography (GLC)


The column used in GLC is coiled and very thin. It is packed with an inert
powder which is coated with a nonvolatile oil. The column is in an oven
which is maintained at a constant, pre-set temperature. The sample analysed
must be in the vapour phase at the temperature at which the column is
operated.

Chromatographic methods of separation

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Components of the mixture analysed are identified by either their retention


times or by spectroscopic analysis. The retention times of the components
are calibrated using known pure substances under identical, specified
conditions. The retention time is the time taken for a component to be
detected after the sample has been injected into the column.
The chromatogram from GLC shows not only the retention time of a
substance but also its concentration which is related to the height and width
of its peak.

Applications of chromatographic methods


1.
2.
3.

Pesticide analysis
Forensic testing
Purification of natural products

END OF CHROMATOGRAPHIC METHODS OF SEPARATION


Practice Question

Chromatographic methods of separation

page 6 of 6

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