2. Adsorption Chromatography
Separation based on their adsorption onto the surface
of solid (stationary phase).
A solid which is insoluble in the solvent chosen may
be used as the fixed phase.
The mode of interaction between the components of
the mixture and the fixed phase is adsorption, Hence
the method is called adsorption chromatography.
Ex; Column chromatography, TLC etc.,
3. Partition chromatography
Partition chromatography, is a chromatographic
technique in which the solute is separated based
on their partition between a liquid mobile phase
and a liquid stationary phase coated on a solid
support.
The support material used in partition
chromatography is usually silica
4. Fall, 2008 4
Thin-Layer Chromatography
Here the mobile phase is a liquid
Flowing past a thin layer of powder on a solid
support.
Substances that are less attracted to the solid or are
more soluble in the liquid move faster
Stationary phase
• glass or plastic plates coated with thin layer of
adsorbent
• Silica gel, alumina, cellulose
Mobile phase
• Solvent or mixture of solvents
5. Thin layer chromatography
• It involves the separation of substances of a mixture over a thin
layer of an adsorbent.
• A thin layer(about 0.2mm thick) of an adsorbent (silica gel or
alumina) is spread over a glass plate of suitable size. The plate
is known as thin layer chromatography plate.
• The solution of the mixture to be separated is applied as a
small spot about 2 cm above one end of the TLC plate.
• The glass plate is then placed in closed jar containing the
solvent (Below 2cm height).
• As the solvent in the jar moves up the plate, the components of
mixture move up the plate to different distances, depending on
this degree of adsorption separation takes place.
6. The relative adsorption of each component of the
mixture is expressed in terms of its retention factor
i.e., Rf
value
7. Rf value
Rf values are used in identification of each of the
component.
• The retention factor, or Rf, is defined as the distance traveled by
the compound divided by the distance traveled by the solvent
• For example, if a compound travels 2.1 cm and the solvent front
travels 2.8 cm, the Rf
is 0.75:
8. Paper Chromatography
• In this technique, the stationary phase is considered
to be the cellulose network of the paper.
• The mobile phase known as the developing solvent
consists of either one solvent or a mixture of
different solvents.
9. Paper Chromatography
• In this the mixture of compounds is applied on the
paper as a spot little above the lower end and then
this end is dipped in the solvent. When the solvent
has risen more than two third length of the paper,
then it is removed from the solvent. The paper is
dried and is known as chromatogram.
• Now, the spots of different compounds can be
visualised using some suitable chemicals.
10. Rf value
The ratio of the distance travelled by the compound in
a particular solvent to that the distance travelled by
the solvent is a constant and is known as retention
factor (Rf).
For example, if a compound travels 2.1 cm and the
solvent front travels 2.8 cm, the Rf
is 0.75:
12. Ion Exchange Chromatography
• Ion exchange chromatography is a special name
given to column chromatography when the
stationary phase is an ion exchange resin.
• Synthetic ion exchange resins are high molecular
weight polymeric materials containing large
number of ionic functional groups per molecule.
Cation exchangers contain sulphonic acid groups
(RSO3
–
H+
) or carboxylic acid groups (RCOO–
H+
). Anion
exchange resin contains amines attached to the
polymer molecule RN(CH2)3
+
OH–
).
13. Ion Exchange Chromatography
• In ion exchange chromatography a column is packed with an
acid resin and treated first with hydrochloric acid to make sure
that all exchange points were occupied by hydrogen ions.
• A mixture of rare earths as their chlorides is sent down the
column. This resulted in the displacement of hydrogen ions by
rare earth cations.
• The rare earth ions could then be eluted one after another. Since
elution with water was very slow, a solution of citric acid was
used as eluting solvent.
• The cations moved at different rates depending on the stability
of the corresponding complex with citric acid.
15. Gas Chromatography (GC)Gas Chromatography (GC)
GC is currently one of the most popular methods for
separating and analyzing compounds.
This is due to its high resolution, low limits of
detection, speed, accuracy and reproducibility.
GC can be applied to the separation of any
compound that is either naturally volatile (i.e., readily
goes into the gas phase) or can be converted to a
volatile derivative.
A simple GC system consists of:
. G 1. Gas source
2. Injector or sample application system
3. Chromatographic column
4. Detector & computer or recorder
17. Gas sourceGas source
• It provides all the necessary gas supplies.
• The most widely used gases are H, He, N2 and air.
InjectorInjector
• We use a syringesyringe to inject sample onto the column.
• It is situated inside a thermostaticallythermostatically controlled
enclosure.
18. ColumnColumn
• It contains the column and an oven.
• The column is the essential device to
achieve the necessary separation.
• The oven is used to control the column
temperature.
• The column has two kinds:
Packed columnPacked column Capillary columnCapillary column
19.
20. DetectorDetector
• There are wide range of detectors available
each having unique operating parameters and
its own performing characteristics.
• The output of detector is electronically modified.
21. Types of detectorsTypes of detectors
• The two types of detectors are
TCD:TCD: Thermal Conductivity DetectorThermal Conductivity Detector
ECD:ECD: Electron Capture DetectorElectron Capture Detector
22. TCD Detector
A TCD detector consists of an electrically-heated wire.
The temperature of the sensing element depends on the
thermal conductivity of the gas flowing around it.
Changes in thermal conductivity, such as when organic
molecules displace some of the carrier gas, cause a
temperature rise in the element which is sensed as a
change in resistance.
The TCD is not as sensitive as other detectors but it is
non-specific and non-destructive.
23. ECD Detector
Uses a radiactive Beta emitter
(electrons) to ionize some of the
carrier gas and produces a
current between a biased pair of
electrodes.
When an organic molecule that
contains electronegative
functional groups, such as
halogens, phosphorous and nitro
groups, pass by the detector,
they capture some of the
electrons and reduce the current.
