Chromatography-Dr Bills

CHROMATOGRAPH
Y
II
Chromatography
Is a technique used to separate and identify
the components of a mixture.
Works by allowing the molecules present in

the mixture to distribute themselves between
a stationary and a mobile phase.
Molecules that spend most of their time in the

mobile phase are carried along faster.
Classification of chromatography
according to mobile phase:
1- Liquid chromatography: mobile phase is a

liquid. (LLC, LSC).
2- Gas chromatography : mobile phase is a gas.
(GSC, GLC).
Classification according to the packing
of the stationary phase:
1- Thin layer chromatography (TLC): the stationary
phase is a thin layer supported on glass, plastic or
aluminium plates.
2- Paper chromatography (PC): the stationary phase is
a thin film of liquid supported on an inert support.
3- Column chromatography (CC): stationary phase is
packed in a glass column.
Classification according to the force of
separation:
1- Adsorption chromatography.
2- Partition chromatography.
3- Ion exchange chromatography.
4- Gel filtration chromatography.
5- Affinity chromatography.
Different Types of chromatography
Mechanism Mobile phase Stationary phase Mode or type
Solutes move at different rates according to the forces Liquid or gas Solid that attracts the solutes Adsorption
.of attraction to the stationary phase Chromatography
Solutes equilibrate between the 2 phases according to Liquid or gas Thin film of liquid formed on Partition
their partition coefficients the surface of a solid inert Chromatography
support
Solute ions of charge opposite to the fixed ions are Liquid Solid resin that carries fixed Ion Exchange
attracted to the resin by electrostatic forces & replace containing ions & mobile couterions of Chromatography
.the mobile counterions electrolytes opposite charge attached by
covalent bonds
:Molecules separate according to their size Liquid Porous gel with no attractive Molecular Exclusion
1.Smaller molecules enter the pores of the gel, and action on solute molecules Chromatography
need a larger volume of eluent.
2.Larger molecules pass through the column at a
faster rate.
Special kind of solute molecules interact with those Liquid or gas Solid on which specific Affinity
immobilized on the stationary phase molecules are immobilized Chromatography
Columnar Chromatography (CC)
This includes chromatographic methods in which:
The stationary phase is packed into a column.
The mobile phase is a moving liquid or gas.
According to the mechanism of separation of solutes,

five major types of CC are ditinguished. Usually, one
mechanism predominates but does not exclude the
others
Column Chromatography
Column chromatography
Stationary phase is held in a
narrow tube through
which the mobile phase is
forced under pressure or
under the effect of gravity
Definition Term
Mobile liquid phase with no affinity to the stationary phase
Solvent
.(i.e. inert towards it) & no effect on solutes
Any liquid with more affinity to the stationary phase than
the solvent but less than solutes and just capable to move Developer
.them through the column
.Any liquid that passes out of the column Effluent
Any liquid that has lesser affinity to the stationary phase
Eluent
.than solutes but is capable to move them out of the column
.Fraction of eluent containing a required specific substance Eluate
Volume of mobile phase that passes :)or retardation volume( Retention
.out of the column, before elution of a specific substance volume (VR)
Open Column Chromatography
(Traditional column chromatography)
Traditional CC is characterized
by addition of mobile phase
under atmospheric pressure
and the stationary phase is
packed in a glass column.
Packing & operating the column
1- Packing
The selection of the method of packing depends
mainly on the density of the solid. Techniques
used are the wet, dry & slurry methods.
In all cases avoid inclusion of air bubbles
2- Sample Application
Apply evenly & in a concentrated solution to the

top of the column which is protected from
disturbance (e.g. add glass wool or filter paper).
3. Elution techniques
Procedure Technique
Addition of solvent mixture of fixed composition during the Isocratic
.whole process elution
Continuous or linear elution: in which there is continuous Gradient

change in the composition of the mobile phase over a period of elution
.time (e.g. polarity, pH or ionic strength)
Step wise or fractional elution: in which the change is not

continuous i.e. a sudden change in the composition of the
mobile phase is followed by a period where the mobile phase is
.held constant
4- Detection
On-column detection for colored or fluorescent
compounds directly after developing the
chromatogram.
Monitoring of eluted fractions (PC or TLC).
Using special detectors connected to the column such

as refractive index, UV detectors, etc…
Factors affecting solutes separation in CC
( Factors affecting column efficiency)
Effect Factor
Decrease of size improves separation (but very small particles need Particle size of solid stationary phase
.high pressure) (or of support)
.Efficiency increases as ratio length / width increases Column dimensions
Non uniform packing results in irregular movement of solutes
through column & less uniform zone formation, (i.e. band broadning Uniformity of packing
.or tailing)
Increase in column temperature results in speed of elution but does
Column temperature
.not improve separation (tailing)
Solvents should be of low viscosity (to give efficient resolution) & h

Eluting solvent
.igh volatility (to get rapid recovery of the substances)
.Uniform & low flow rate gives better resolution Solvent flow rate
Discontinuous flow disturbs resolution Continuity of flow
.Deactivation of adsorbent decreases separation Condition of adsorbent
.Substances of high concentration move slowly Concentration of solutes
Thin layer chromatography (TLC)
TLC is a method for identifying substances and
testing the purity of compounds.
TLC is a useful technique because it is relatively

quick and requires small quantities of
material.
Separations in TLC involve distributing a mixture of two or more
substances between a stationary phase and a mobile phase.
The stationary phase:

is a thin layer of adsorbent (usually silica gel or alumina) coated on a
plate.
The mobile phase:

is a developing liquid which travels up the stationary phase, carrying the
samples with it.
Components of the samples will separate on the stationary phase
according to
how much they adsorb on the stationary phase versus how much they
dissolve in the mobile phase.
Thin Layer Chromatography (TLC)
TLC
Preparing the Chamber
To a jar with a tight-fitting lid add enough of the
appropriate developing liquid so that it is 0.5 to
1 cm deep in the bottom of the jar.
Close the jar tightly, and let it stand for about 30

minutes so that the atmosphere in the jar
becomes saturated with solvent.
Preparing the Plates for Development
With a pencil, etch small notches into the adsorbent about 2
cm from the bottom of the plate.
The notches should be on the edges of the plate, and each
notch should be the same distance up from the bottom of
the plate.
The notches must be farther from the bottom of the plate
than the depth of the solvent in the jar.
Using a drawn-out capillary tube, spot the samples on the
plate so that they line up with the notches you etched.
Question: What is wrong with the plate shown
below?
Developing the Plates
After preparing the development chamber and
spotting the samples, the plates are ready for
development.
Be careful to handle the plates only by their edges, and
try to leave the development chamber uncovered for
as little time as possible.
When the plates are removed from the chamber,
quickly trace the solvent front (the highest solvent
level on the plate) with a pencil.
Identifying the Spots (visualization)
If the spots can be seen, outline them with
a pencil.
If no spots are obvious, the most common
visualization technique is to hold the
plate under a UV lamp.
Many organic compounds can be seen
using this technique, and many
commercially made plates often
contain a substance which aids in the
visualization of compounds.
Visualizing Agents
General Visualizing agents: H2SO4

Alkaloids: Dragendorff’s reagent
Cardiac glycosides: Antimony trichloride
Sugar: Aniline phthalate
Amino acids: Ninhydrin
Interpreting the Data
The Rf (retention factor) value for each spot
should be calculated.
It is characteristic for any given compound on
the same stationary phase using the same
mobile phase for development of the plates.
Hence, known Rf values can be compared to
those of unknown substances to aid in their
identifications.
Retention Factor (RF)
(Note: Rf values often depend on the temperature and the solvent used in
the TLC experiment.
the most effective way to identify a compound is to spot known

substances – authentic - next to unknown substances on the same
plate.)
In addition, the purity of a sample may be estimated from the

chromatogram.
An impure sample will often develop as two or more spots, while a

pure sample will show only one spot
Summary
A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a
solid adsorbent (usually silica or alumina).
A small amount of the mixture to be analyzed is spotted near the bottom of this plate.
The TLC plate is then placed in a shallow pool of a solvent in a developing chamber
so that only the very bottom of the plate is in the liquid.
This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by
capillary action.
As the solvent moves past the spot that was applied, an equilibrium is established for
each component of the mixture between the molecules of that component which
are adsorbed on the solid and the molecules which are in solution.
In principle, the components will differ in solubility and in the
strength of their adsorption to the adsorbent and some
components will be carried farther up the plate than others.
When the solvent has reached the top of the plate, the plate is
removed from the developing chamber, dried, and the
separated components of the mixture are visualized.
If the compounds are colored, visualization is straightforward.

Usually the compounds are not colored, so a UV lamp is
used to visualize the plates.
Watch on
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Column and Thin Layer
Chromatography?
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CHROMATOGRAPH

Works by allowing the molecules present in

Molecules that spend most of their time in the

1- Liquid chromatography: mobile phase is a

According to the mechanism of separation of solutes,

Apply evenly & in a concentrated solution to the

Continuous or linear elution: in which there is continuous Gradient

Step wise or fractional elution: in which the change is not

Monitoring of eluted fractions (PC or TLC).

Using special detectors connected to the column such

Solvents should be of low viscosity (to give efficient resolution) & h

TLC is a useful technique because it is relatively

The stationary phase:

The mobile phase:

Close the jar tightly, and let it stand for about 30

General Visualizing agents: H2SO4

the most effective way to identify a compound is to spot known

In addition, the purity of a sample may be estimated from the

An impure sample will often develop as two or more spots, while a

If the compounds are colored, visualization is straightforward.

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