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Dept. of Chemistry, University of the
West Indies MonaC.A.P.E. CHEMISTRY WORKSHOP
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What is Chromatography?
Recall the first time you were introduced to colour in art
class and you learnt of the three primary colours and the
resulting secondary colours.
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Chromatography is concerned with being able to separate
mixtures into their individual components.
This can be useful for detection purposes, but may also be
used to quantify the different components.
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Types of chromatography
There are many types, however, you need to be familiar
with the following
1. Paper
2. Thin layer (tlc)
3. Column
4. Gas-liquid (glc)
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Principles of Chromatography
All types of chromatography have the following principles
in common.
1. There are two phases: the stationary phase and the
mobile phase
2. The separation of the mixture is due to differing
interaction of the components with the stationary phase
3. The two mechanisms which can occur are partitioning
and adsorption
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What is the stationary phase?
This is the immovable phase and may either be a solidsupport:
Cellulose in the paper
Silica or alumina
or it may be a non-volatile, viscous liquid coated unto a
solid surface
A long-chain alkane of high boiling point on a SiO2
support
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What is the mobile phase?
This is the solvent which moves through the column or
over the surface of the stationary phase and can be a
gas or a liquid.
There are different types of solvents, ranging from polar
(such as water) to non-polar (such as alkanes)
The mobile phase can be a mixture of solvents, in which
case the ratio is quoted
e.g. methanol:water (2:1)
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PARTITIONING AND ADSORPTION
Adsorption chromatography occurs when the componentsof the mixture (solute molecules) are bound to the surface
of the stationary phase.
The stationary phase is a polar solid (such as silica or
alumina) and the polar solute molecules are bound to this
surface. The more polar the solute, the more strongly
bound it is to the stationary phase
As the mobile phase passes over this, the solute
molecules are eluted, from least polar to most polar in
turn.
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Partitioning occurs between liquids.
If colourless chloroform is added to aqueous iodine (a
brown solution,) two layers develop, with the chloroform
layer below the aqueous layer and having a purple colour.
The iodine moves between the two layers until it reaches
an equilibrium, at which point it is in a definite ratio. This is
partitioning.
If both the stationary and the mobile phases are fluids, then
the solute will be partitioned between the two.
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Solute in the mobile phase will move along with it and
elution will take place starting with those compounds
more soluble in the mobile phase and ending with thoseleast soluble.
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PAPER CHROMATOGRAPHY
Filter paper is used to aid in separation
Cellulose fibres contain water which acts as the stationary
phase.
A small dot of mixture is placed on the paper, which is
placed in a jar containing a shallow layer of solvent and issealed
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The Process of Paper
Chromatography
https://defra.jot.com/System/TmpImageUpload/Chromatography.jpg
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Separation of black ink using Paper Chromatography
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Retention factor (also called
retardation factor) : Rf
The movement of a soluterelative to the movement of
the solvent front
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THIN LAYER CHROMATOGRAPHY
Stationary phase is a thin layer of silica (SiO2) or alumina
(Al2O3) coated unto a plastic or glass support.
Setup is similar to that of paper chromatography, but thisutilises adsorption and not partition.
Useful for separating colourless components which can bedetected by use of appropriate chemicals or techniques
(visualising agents)
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Thin layer chromatography used to
separate the components of chlorophyll
http://en.wikipedia.org/wiki/Chromatography
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Visualising agents
1. Iodine crystals may be
added to the solvent andas iodine vapour
evolves, this is
accumulated on the
spots of the separated
solute.
Result is dark brown
spots on a yellow
background
http://www.chem.ucsb.edu/~kalju/chem110L/public/TLC_2004.png
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1. Spraying amino acids with ninhydrin causes them to
appear lilac in colour
http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html
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1. Visualising colourless solute using UV light
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Advantages of TLC over Paper Chromatography
1. TLC is faster (approximately three times as fast)2. TLC works well with very small samples
3. The thin layer can be made from different solids, so a
wide range of mixtures can be separated by carefully
selecting the mobile and stationary phases
4. TLC can be used to quickly select the best conditions for
larger scale separation (such as by column
chromatography)
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Application of TLC
Wide range of applications1. Pesticide analysis: separation of chlorinated insecticides
2. Chemical Forensics: separation and detection of
alkaloids (e.g. morphine and opium) and cannibis also
used as a screen for explosives
3. Drug testing: Detection of steroids
4. Pharmaceutical: Determination of the purity of new drugs
(quality control purposes)
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COLUMN CHROMATOGRAPHY
Stationary phase is silica (SiO2) or alumina (Al2O3) placedin a column. (note, this is a polar phase)
The mixture is placed above the stationary phase andeluted with the mobile phase under gravity or under
pressure (flash chromatography.)
Can be used to separate quantities ranging from
micrograms to kilograms and is based on adsorption
mechanism
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The aim is to achieve good separation by carefully
choosing solvent systems which allow one component to
move through the column faster than the othercomponents.
Stationary phase is polar and will bind strongest to the
most polar component of the mixture
Mobile phase ranges from non-polar through to polar, with
a different solvent mixture being used to elute successivelypolar components
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http://www.chemguide.co.uk/analysis/chromatography/column.html
Useful for purification purposes as mixtures can be separated into individual
components and be collected in fractions.
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Application of Column Chromatography
1. Purification of natural products2. Purification of pharmaceuticals
3. Purification of chemical reactions
4. Separation of dyes and pigments
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GAS-LIQUID CHROMATOGRAPHY
Used to separate and identify small quantities of gases, liquidsand volatile solids
Vapourised sample is carried by a mobile phase (inert gas)over a stationary phase (non-volatile liquid on a solid support)
Partitioning of the components of the sample occurs and they
move through the column based on
their volatility
their relative solubility in the mobile and stationary phases
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Diagram of a typical Gas-Liquid Chromatograph
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The components leave the column after a definite time
period.
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Retention time: time between the injection of the mixture
and the centre of the peak corresponding to a component
Area under a component peak is proportional to the
amount of that component in the mixture
Useful for detection, determination and quantification of
compounds and is used in conjunction with mass
spectrometry for confirmation of the components eluted.
(GC-MS)
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Application of GLC
1. Pesticide analysis2. Analysis of crude oil
3. Environmental analysis: detection of pollutants
4. Determination of the components of natural oils andflavours
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1. A particular component with a low affinity for the stationary phase will
generally move a ________ distance along the stationary phase than a
component with a high affinity for the stationary phase in a
chromatography experiment.
A. longerB. shorter
2. Let Ds be the distance the solvent travels, and let D1 be the distance
component 1 travels on the chromatogram. The retention factor for
component 1 is defined to be:
A. D1 x Ds
B. D1/Ds
C. 1/( D1 x Ds)
3. If a beaker is used as a "developing jar" in an experiment. When the
TLC plate is set in this beaker, the solvent in the beaker must be:
A. above the pencil line used to guide the spotting of samples
B. deep enough to cover the entire TLC plate
C. deep enough to come about halfway up the TLC plate
D. below the pencil line used to guide the spotting of samples