Chromatography
Chromatography
Chromatography
Chromatography
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Chromatography
Chromatography
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Definition (IUPAC):
Mikhail Tsvet
Chromatography is a physical method of separation in
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which the components to be separated are distributed
between two phases, one of which is stationary while the
other moves in a definite direction.
Chromatography
Chromatography
Chroma: Color Graphein: To write
Definition (IUPAC):
Chromatography is a physical method of separation in
which the components to be separated are distributed
between two phases, one of which is stationary while the
other moves in a definite direction.
Types of Chromatography
Basic concepts of chromatography
Chromatography is a separation technique where component
molecules (solutes) in a sample mixture are transported by a mobile
phase over a stationary phase.
The mobile phase may be a gas or a liquid (solvent system) and the
stationary phase may be a liquid film on the surface of an inert
support material or a solid surface.
The solute, mobile phase and stationary phase form a ternary system.
Interaction occurs between the solute and stationary phase so that the
solute is distributed between the stationary phase and mobile phase.
Attraction of the solute for the stationary phase results in retardation
of its movement through the chromatography system. Different
components (solutes) will move at differing rates since each will have
a slightly different affinity for the stationary phase with respect to the
mobile phase.
Each component or solute (A, B, C) is distributed between the two
phases with an equilibrium established defined by the distribution
ratio (previously known as the partition ratio); thus for component
Basic concepts of chromatography
Thin layer chromatography (TLC)
Retention factor
Thin layer chromatography (TLC)
Column chromatography
Adsorbent
Activated alumina (Al2O3) and silica gel
(SiO2) are the most commonly used and
commercially available adsorbents in
the column chromatography
procedure. Activated alumina and
charcoal are strong adsorbents.
Calcium carbonate, calcium phosphate,
magnesia, and shake lime are
examples of intermediate adsorbent
materials.
Column chromatography
Adsorbent
Activated alumina (Al2O3) and silica gel (SiO2) are
the most commonly used and commercially
available adsorbents in the column
chromatography procedure. Activated alumina
and charcoal are strong adsorbents.
Calcium carbonate, calcium phosphate,
magnesia, and shake lime are examples of
intermediate adsorbent materials.
The absorbability of organic compounds depends
on the nature and number of the polar groups
present in the particular compounds.
Column chromatography
Characteristics of Adsorbent
Adsorbent particles have uniform shapes and
sizes in the range of 60-200 μm in diameter.
The adsorbent has high mechanical stability and
chemical inertness.
The compound itself is colourless, inexpensive,
and readily available.
It has free passage for the mobile phase.
It should be suitable for a wide range of samples.
Column chromatography
Column Chromatography Solvent Selection:
• Adsorption depends on the selection of solvent in the column chromatography
procedure. A relatively nonpolar solvent is used for placing the solute in a
column but a polar solvent is used for developing a chromatogram.
• The polarity of the solvent gradually increases by eluting absorbed material. If
the compounds are eluted rapidly, poor separation is obtained. We used milder
absorbent and slightly polar solvent for the slow elution procedure. Therefore,
we select nonpolar solvents for weakly absorbed compounds and polar solvents
for strongly absorbed compounds in the column chromatography procedure.
Polarity trends:
Petroleum ether < carbon tetrachloride < cyclohexane < carbon disulfide < ether <
acetone < benzene < ethyl acetate < chloroform < ethanol < methanol < water <
pyridine < acetic acid.
Column chromatography
Sample Preparation
• The sample should be applied to the top of the column as evenly as possible,
in as concentrated a solution of the eluting solvent as possible, avoiding
disturbance of the column packing.
• The top of the column can be protected with a thin layer of sand, glass wool, filter
paper or ballotini beads.
• When all of the sample has been adsorbed, the void can be filled with solvent and
the chromatogram developed.
• The supply of solvent can be replenished as required.
Column chromatography
lsocratic:
Allowing a solvent mixture of unvarying
composition to run through the column
until separation is complete.
Fractional elution:
Liquid phase • Using several different solvents of gradually
chromatography increasing strength for the successive desorption of
on open columns Different components. This is known as stepwise
elution.
• It has the advantage that sharper
separations may be obtained than if only one
strongly eluting solvent, capable of moving even
Gradient elution: the most firmly bound of the components
• It involves the use of a continuously changing eluting medium.
of the mixture.
The effect of this gradient is to elute successively • One con of this technique, however, is that a
the more strongly adsorbed substances and at the same time to given compound may give rise to more than one
reduce tailing. This means that the chromatographic bands will peak by appearing in the eluates of successive
tend to be more concentrated and thus occupy less of the column. steps.
Column chromatography
Adsorption chromatography
Liquid phase
chromatography Partition column chromatography
on open columns
Ion exchange chromatography
Column chromatography
Adsorption chromatography:
Adsorbent:
1. The lattice of the common porous adsorbents, e.g. alumina and silica, is terminated at the surface with polar hydroxyl
groups, and it is these groups which provide the means for the surface interactions with solute molecules.
Elution:
2. The eluant systems used in adsorption chromatography are based on nonpolar solvents, commonly hexane, containing a
small amount of a polar additive, such as 2-propanol.
3. When the sample is applied, solute molecules with polar functionality will bond to the active sites on the packing; they
will subsequently be displaced by the polar modifier molecules of the eluant as the chromatogram is developed, and will
pass down the column to be re-adsorbed on fresh sites.
4. The ease of displacement of solute molecules will depend on their relative polarities. More polar molecules will be
adsorbed more strongly, and hence will elute more slowly from the column.
Column chromatography
Adsorption chromatography:
Solvent:
Polarity trends:
pure water > methanol > ethanol > propanol > acetone > ethyl acetate > diethyl ether > chloroform > dichloromethane >
benzene > toluene > trichloroethylene > carbon tetrachloride > cyclohexane > hexane
With the aid of the above series it is possible to select a solvent or solvent mixture with the appropriate eluting power.
Partition column chromatography:
1. Partition column chromatography is a technique that separates a mixture into two liquid phases using a column. The
separation is based on the difference in partition coefficients between the phases.
2. The stationary phase is supported on a solid which is inert to the substances to be separated. The coated solid is packed into
columns as in adsorption chromatography.
Column chromatography
Column chromatography
Ion exchange chromatography:
Ion exchange is a process wherein a solution of an electrolyte is brought into contact with an ion exchange resin and active
ions on the resin are replaced by ions (ionic species) of similar charge from the analyte solution.
• The solution that enters the column is called the influent.
• The solution that exits the column is called the effluent.
• The column uses a stationary phase with a charge opposite to the charged ions
• in the solution.
• The charged ions are attracted to the stationary phase and retained.
• The ions are separated based on their affinity for the ion exchanger.
Column chromatography
Column chromatography
The affinity of cations for the resin in dilute aqueous solution varies mainly as their charge and increases with it; for cations
of the same charge the affinity is inversely proportional to the radius of the hydrated ion. This may be explained in terms of
the polarising power of the cation, i.e. the greater the polarising power of the cation, the greater its affinity for the
resin and the more strongly will it be retained. (The polarising power of a cation is proportional to Z 2 /r where Z is the
charge on the ion and r is the radius of the hydrated ion.)
In dilute solution the affinity of anions for an anion exchange resin depends on the degree of polarisation of the anion by
the exchanger cation; i.e. the greater the degree of polarisation the more firmly shall the anion be held; and the greater
the affinity of the anion for the resin.
Column chromatography
