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SEPARATION TECHNIQUES
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A component is sublimable if it goes
directly to the gaseous phase from the solid phase after heating
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The procedure for caffeine separation, involves heating the powdered
plant material in a wide-mouth container, the sublimable constituent
(caffeine) is first vaporized and collected separately from other constituents
of the mixture
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Fractional distillation
This method is used for the separation of volatile component of a mixture,
having different boiling points
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3
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The mixture is first slightly acidified and shaken with a strong organic
solvent (e
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Chloroform)
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The alkaloid component in the mixture is precipitated out
depending on their degree of basicity
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Fractional Crystallization
This technique exploits the differences in solubility of the components of the
mixture in a particular solvent
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The most soluble component
crystalizes out last
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Hence the literal meaning of Chromatography is “COLOR WRITING”
Definition
Chromatography can be defined as a laboratory technique used for
separating components of a mixture, in which the various components to be
separated are distributed between two phases
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The components of the mixture redistribute themselves between these
two phases
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Chromatography is probably the most powerful and versatile technique
available to the modern analyst
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HISTORY OF CHROMATOGRAPHY
The first scientist to recognize chromatography as an efficient method of
separation was the Russian botanist MIKHAIL TSWETT in 1903
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This
technique was later named Liquid- Solid adsorption Chromatography
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The technique as described by Tswett was largely ignored until the
late 1930‟s and early 1940‟s when Martin and Synge introduced liquidliquid chromatography, they further recommended replacing the liquid

mobile phase, by a suitable gas, hence the concept of gas chromatography
was created
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These forces can be strong
hydrogen bonds, polarity, weak van der-waal forces, acid-base bonding
etc
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k) Mobile phase
This is the solvent that moves the solute (components) through the
stationary phase also called the Eluent

l) Stationary Phase
The Chromatographically retentive immobile phase involved in
chromatographic processes usually solid, but can be liquid as well
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Solubility:
This is the tendency of a component to go into solution
2
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3
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Polarity:
This is the ability of a molecule to possess separate partial positive charge
and a partial negative charge, resulting in a phenomenon called „A DIPOLE
MOMENT‟
The polarity of the mobile phase against that of the stationary phase
plays a very important role in separation
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In chromatography if the stationary phase is polar, the mobile phase in general
should be made non polar, especially in Adsorption chromatography or Partition
chromatography; this is called NORMAL PHASE Chromatography
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5
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The polarity of
the solvent is a factor that affects separation
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Oxygenated organic compounds such as alcohol, ketones, esters e
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c,
possess less strong dipole and are also less H-bonded and are therefore less
polar than H20
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g
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t
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This measures their eluting
power
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Hexane, Petroleum ether
2
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Tetrachloromethane (Benziform)
4
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Toluene
6
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Ethylacetate
8
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Propanol
10
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Methanol
12
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Adsorbent:
Adsorbent can be polar or non- polar
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Silica gel is an example of a polar adsorbent
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When silica gel is polar, the chromatographic technique is referred to as normal
phase chromatography
Other adsorbent used in chromatography include;




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Alumina (Al203) [Aluminum oxide]
Cellulose powder
Kieselgur (Diatomaceous earth)
Starch
Polyamide

CLASSIFICATION OF CHROMATOGRAPHY
Chromatography can be classified on the following basis;
A
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C
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The shape of the solid support/ technique
The nature of the mobile phase
The mechanism of separation
The Application

A
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The nature of the mobile phase
Liquid chromatography
Gas chromatography
C
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The Application
Analytical tool
Preparative tool
Adsorption Chromatography
This separation mechanism depends upon the ability of the solute molecule or
component of a mixture to adhere to a solid support
...
These forces of attraction include; hydrogen bonds, vander Waal
forces etc
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For a polar stationary phase, non-polar components of the mixture spend more time
in the mobile phase than polar compounds
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The mechanism behind this type of chromatography is the formation of a thin film
of liquid on the solid support, which then serves as the stationary phase
...
In other words the
factor that will determine if a component of a mixture will remain in the stationary
phase or move with the mobile phase will be its solubility in the two phases
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Solutes which are more soluble in the mobile phase will move faster than those
that are less soluble
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This type of
chromatography lacks an attractive interaction between the stationary phase and
components of the mixture
...
The pores are normally small and excludes (does
not allow) the larger solute from entering the pores, hence the name Size exclusion
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Gel
filtration is mostly used to separate proteins of different molecular weight and size
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In this type of chromatography, a resin is used as the stationary
phase, this resins are referred to as ion exchange resins
...
Ion exchange resins are
fabrications from an organic polymer substrate
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Trapping of ions takes place
simultaneously with release of another ion of like charge
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Counter-ions of opposite charge are loosely complexed with these groups of
charges that are already present on the resin called the fixed charges
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THE DEVELOPMENT PROCESS
This is the process whereby the components of a mixture move through the
chromatographic system to the point at which they are removed from the system
There are three types of chromatographic development:
 Frontal analysis
 Displacement development
 Elution development

Frontal Analysis
This type of chromatographic development is rarely used, and it‟s probably of
academic interest only
...
In
frontal analysis, the mixture is fed continuously onto the column in the mobile
phase
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Displacement Development
Displacement development is only effective with a solid stationary phase, where
the components of the mixture are adsorbed on its surface
...
Consequently the
components of the mixture must be displaced by a substance more strongly held
than any of the solutes
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The displacer (solvent) is added after the development, where it displaces
the least held component first, before displacing the most strongly held component
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Elution Development
This is the most used method of chromatographic development, even though this
process can only be performed on a column (Partition Chromatography)
In elution development the principle of Partitioning plays a more important role
than adsorption
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Column Chromatography
Column chromatography is used for the separation of large amounts of mixtures
mostly for preparative work
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NB: in column chromatography the mobile phase can be a single solvent or a
mixture of two or more solvents
The stationary phase is placed in a cylindrical tube made of glass closed from the
bottom with a valve
...
The
mobile phase for the separation is then added continuously until equilibrium is
attained
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Slurry packing
A slurry of the adsorbent is made in the mobile phase to be used for the separation,
and then introduced into the column, until an even surface of adsorbent is obtained
Wet packing
An appropriate volume of the solvent is placed in the column, using a funnel and
then the adsorbent is gradually introduced into the column, with constant tapping
to ensure uniformity of adsorbent at the top
Note that in all three methods of packing, the solvent or mobile phase should
be at the top of the adsorbent
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The mobile phase cause a
gradual downward migration of the components of the mixture, and the bands are
separated and subsequently collected
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They
are intended to support the adsorbent and are designed to permit control of solvent
input and effluent collection
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Flow Rate in Column Chromatography
This may be less rapid when compared with other chromatographic techniques like
thin layer chromatography
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There are no rigid rules about flow
rate but usually slower flow rates produces good elution patterns
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The nature of the mobile phase
affects the flow rate

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