2. Chromatography
Chromatography is based on the Greek word
chroma, for colour. This technique is
extensively used to separate mixtures into
their components, purify compounds and also
to test its purity.
Chromatography works on the same principle
as extraction, but one phase remains
stationary and the other flows past.
3. Chromatography
The stationary phase is the phase that stays in
place inside the column and is usually viscous liquid
chemically bonded to the inside of a capillary column
or onto the surfacee of solid particles packed into the
column.
The mobile phase is the solvent moving through
the column and is either liquid or gas.
Fluid entering the column is the eluent, while fluid
leaving the column is the eluate.
Elution is the process of passing liquid or gas
4.
5. Adsorption Chromatography
Adsorption chromatography is a process
of separation of components in a mixture introduced
into chromatography system based on the relative
differences in adsorption of components to the
stationary phase present in the
chromatography column.
This adsorption chromatography applies to only solid-
liquid or solid-gas chromatography. Because the
adsorption phenomenon is inherent property of solids
and hence it is used with only solid stationary phase
chromatographies.
8. Adsorption Chromatography
1. Column Chromatography
It involves the separation of a mixture over a column of adsorbent (stationary phase)
packed in a glass tube.
The mixture adsorbed on adsorbent is placed on the top of the adsorbent column
packed in a glass tube.
An appropriate eluant which is a liquid is allowed to flow down the column slowly
The most readily adsorbed substance are retained near the top and the others come
down to various distance in the column
9. 1. _
2. Thin Layer Chromatography
This is separation of substances of a mixture over a thin layer of an adsorbent coated on
glass plate.
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 or chromaphate.
The solution of the mixture to be separated is applied as a small spot about 2cm above
one end of the TLC plate.
10. Partition Chromatography
Definition:
This form of chromatography is based on a thin film
formed on the surface of a solid support by a liquid
stationary phase. Solute equilibrates between the
mobile phase and the stationary liquid.
11. Partition Chromatography
Partition chromatography is process of
separation whereby the components of the
mixture get distributed into two liquid phases due
to differences in partition coefficients during the
flow of mobile phase in the chromatography
column.
This mode of partition chromatography applies to
Liquid-liquid, liquid-gas chromatography and not
to solid-gas chromatography. Because partition is
the phenomenon in between a liquid and liquid or
liquid and gas or gas and gas. But not in solid
involvement.
12. Partition Chromatography
Priciple:
Separation of components of a sample mixture occurs
because of partition. Stationary phase is coated with a liquid
which is immiscible in mobile phase.
Partition of component of a sample between sample and
liquid/gas stationary phase retard some components of
sample more as compared to others. This gives basis for
separation.
The stationary phase immobilizes the liquid surface layer,
which becomes stationary phase. Mobile phases passes over
the coated adsorbent and depending upon relative solubility in
the coated liquid, separation occurs. The component of
sample mixture appear separated because of differences in
their partition coefficient.
13. This form of
chromatography is
based on a thin film
formed on the
surface of a solid
support by a liquid
stationary phase.
Solute equilibriates
between the mobile
phase and the
stationary liquid.
14.
15. Gas Liquid Chromatography
A form of chromatography in which the mobile phase
is a gas and the stationary phase is a liquid, usually
on small beads packed in a long column
Used to separate and analyze compounds
Sample has to be able to be vaporized without
decomposition
Based on boiling point/vapor pressure
Typical uses:
Testing purity of particular substance
Separation of different components of a mixture
Prepare pure compounds from a mixture
16. Mobile phase
Inert carrier gas (usually He or N)
Stationary phase
Layer of liquid or polymer on inert solid support
Inside a glass or metal tubing (COLUMN)
17. • Compound is injected with syringe into sample
injector
• Compound is carried by carrier gas and vaporized
• Vaporized sample interacts with walls of column
– Some samples interact more some less
• Due to interaction samples elute at different times
– Retention times
– Comparison of retentions times is what is useful
• A detector monitors the outlet stream from the
column
18.
19.
20. Carrier Gas Pressure Regulator and Flow
Meter
H2 : It has a distinctly better thermal conductivity
and lower density. Demerits are its reactivity with
unsaturated compounds and hazardous explosive
nature
He : It has an excellent thermal conductivity, low
density, inertness and it permits greater flow
rates. It is highly expensive
N2 : It offers reduced sensitivity and is
inexpensive
Air : It is employed only when the atmospheric
O2 is beneficial to the detector separation.
21. Sample Injection System
(a) Liquid Samples : They are usually injected by
hypodermic syringes through a self-sealing silicon-
rubber septum into a preheated-metal-block flash
evaporator. The sample is vapourize as a ‘plug’ and
carried right into the column by the respective carrier
gas. Sample size ranges between 1–10 μl.
(b) Solid Samples : These are either dissolved in volatile
liquids (solvents) or temporarily liquefied by exposure
to infrared heat.
(c) Gas Samples : They are best handled and injected by
gas-light syringes or a gas-sampling valve, usually
termed as a stream-splitter.
22.
23. Separation Column
Types:
Packed
Packed columns contain a finely divided, inert, solid
support material (commonly based on diatomaceous
earth) coated with liquid stationary phase. Most
packed columns are 1.5 - 10m in length and have an
internal diameter of 2 - 4mm.
Capillary a.k.a Open Tubular
Capillary columns have an internal diameter of a few
tenths of a millimeter.
Wall-coated open tubular (WCOT)
Support-coated open tubular (SCOT)
24. Wall-coated columns consist of a capillary
tube whose walls are coated with liquid
stationary phase. In support-coated columns,
the inner wall of the capillary is lined with a
thin layer of support material such as
diatomaceous earth, onto which the stationary
phase has been adsorbed.
SCOT columns are generally less efficient
than WCOT columns.
Both types of capillary column are more
efficient than packed columns.
28. Detector
Types of selectivity:
Non-selective detector - responds to all
compounds except the carrier gas
Selective detector - responds to a range of
compounds with a common physical or chemical
property
Specific detector - responds to a single
chemical compound
29.
30.
31.
32. Liquid-Liquid Chromatography
Components within a mixture are separated in
a column based on each component's affinity
for the mobile phase. So, if the components
are of different polarities and a mobile phase
of a distinct polarity is passed through the
column, one component will migrate through
the column faster than the other. Because
molecules of the same compound will
generally move in groups, the compounds are
separated into distinct bands within the
column. If the components being separated
are colored, their corresponding bands can be
seen.
33.
34. Other varieties of Liquid
Chromatography
Partition Chromatography
Liquid-Solid Chromatography
Ion Exchange or Ion Chromatography
Size Exclusion Chromatograhy
Affinity Chromatography
Chiral Chromatography
Plate Theory or Rate Theory