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Chromatography is the term used to describe a

separation technique in which a mobile phase
carrying a mixture is caused to move in contact
with a selectively absorbent stationary phase.

There are different kinds

of chromatography,
which differ in the
mobile & the stationary
phase used.

Introduction to Chromatography How can the separation be carried

Original Chromatography Experiment In all chromatographic separation, the sample is
transported by a mobile phase
Start: End:
A series of colored
(may be gas, a liquid or
A glass column is
filled with powdered bands is seen to form, a supercritical liquid).
limestone (CaCO3). corresponding to the
different pigments in the
original plant extract. This mobile phase is then forced through an
An EtOH extract of These bands were later immiscible stationary phase (fixed in a place in a
leaf pigments is determined to be column or on a solid surface).
applied to the top of chlorophylls,
xanthophylls & The stationary phase needs to
the column.
EtOH is used to flush carotenoids. be something that does not react
the pigments down with the mobile phase or the
Mikhail Tswett, Russian
the column. Botanist (1901)

How can the separation be carried How can the separation be carried
out..? out..?
Separation process involves interaction between Due to the difference interaction btwn packing
packing material and sample : material & sample separation can be done.
Components of the sample distribute themselves As the consequence of these differences in mobility,
between the mobile and stationary phase to sample components are then separate into discrete
varying degrees. bands or zones, that can be analyzed qualitatively
Those components that are strongly retained by
and/or quantitatively.
the stationary phase move only slowly with the
flow of mobile phase.
In contrast, components that are weakly held by
Retention Time, tR refer to the time
the stationary phase travel rapidly. taken for a solute to elute from a column.


Separation principle for a sample Classification of Chromatography

with two analytes; A and B Techniques

Adsorption Chromatography
A move faster Partition Chromatography
than B because B Ion Exchange and Size Exclusion
interacts strongly Chromatography
with the
stationary phase.

Adsorption Chromatography Partition Chromatography

One of the oldest types of chromatography. The stationary phase is a liquid supported on
It utilizes a liquid or gaseous mobile phase that an inert solid.
is adsorbed onto the surface of a solid The mobile phase may be a
stationary phase. liquid (liquid liquid partition
chromatography) or a gas (gas
liquid chromatography, GLC)
The stationary phase is a solid. The component distribute
The mobile phase may be a liquid between the two phases
(liquid solid chromatograph) or through a combination of
a gas (gas solid chromatography) polar and non polar process.

Ion Exchange and Size Exclusion

Normal Phase Chromatography
A polar stationary phase (e.g., methanol, silica) is
Ion Exchange Chromatography used with non polar mobile phase ( e.g., hexane).
This favors retention of polar compounds and
Used ion exchange resin as the stationary phase. elution of nonpolar compounds.
The mechanism of separation is based on ion
Polarity : RED < YELLOW < GREEN
exchange equilibrium. Sample
Size Exclusion Chromatography RED
Solvated molecules are separated according to
their size by their ability to penetrate a sieve
like structure (the stationary phase).
35 58 63 tR /s


Reversed Phase Chromatography

A non - polar stationary phase (C 18 or n-octyldecyl)
is used with polar mobile phase (water or methanol).
This favors retention of non - polar compounds &
Gas Chromatography
elution of polar compounds.
Polarity : RED < YELLOW < GREEN


35 58 63 tR /s

Introduction to GC Schematic Diagram of GC

One of the most popular instrumental method used
for the separation & identification of chemical
This is due to its high resolution, low limits of
detection, speed, accuracy and reproducibility.
The mobile phase is a gas & the stationary phase
can be either a solid or a non- volatile liquid.
A sample is introduced into a heated injector,
carried through a separating column by an inert gas
and detected as a series of peaks on a recorder
when components leave the column.

Basic Instrumentation of Gas

Gas system Column oven 1. Gas System
Injection system Detector Carrier gas (Inert gas) Helium / Argon /
Column Data system Nitrogen - Chromatographic grade gases (high
O2 is usually avoided since it will oxidize the
stationary phase.
Choice dictated by type of detector, cost,
Need - Pressure regulator for constant inlet
pressure & flow controller for constant flow rate.


Instrumentation Instrumentation
1. Gas System 1. Gas System
Carrier Gas

The mobile phase

or carrier gas flows
through the
instrument from a
pressurized tank.

Two Stage Tank Regulator

Instrumentation Instrumentation

Flow rate
GC Flow Controller 2. Injection system

The flow rate of the gas

(a) Injector port
is controlled by a two
(sample introduction)
stage regulator on the
(b) Syringe Needle / Auto
gas tank and additional
controls within the

Instrumentation Instrumentation
2. Injection system 2. Injection system
The injection port consists of a rubber septum
through which a syringe needle is inserted to inject
the sample.
The injection port is maintained at a higher
temperature than the boiling point of the least
volatile component in the sample mixture.

Manual-Syringe Needle Auto sampler


Instrumentation Instrumentation
2. Injection system 2. Injection system
Enter from Exit to
Purpose of Injection Injector Detector
Deposit the sample into the column
in the narrowest band possible.
The shorter the band at the
beginning of the chromatographic
Packed Column
process-produced tall narrow peaks.
installed in
Gives maximum resolution &
Oven Compartment.
sensitivity. Good Resolution..!
Therefore type of injection method &
operating conditions is critical in
obtaining precise & accurate results.

Instrumentation Instrumentation
2. Injection system 2. Injection system
Samples Samples Injection type : Split Injection
Samples may be pure compounds. Mechanism by which a portion of the injected solution
However, very often prepared as diluted solutions is discarded.
due to the sensitivity of the detection methods. Only a small portion (1/1000 - 1/20) of sample goes
Other methods Solid-phase extraction, Soxhlet through the column.
extraction etc. Used for concentrated samples (> 0.1%)
Sample size will depend on the types of column Can be performed isothermally.
used. Fast injection speed.
Injector & rubber septum contamination does not
usually noticed.

Instrumentation Instrumentation
2. Injection system 2. Injection system
Samples Injection type : Splitless Injection Samples Injection type : On Column Injection

Most of the sample goes through the column (85 - All of the sample is transferred to the column
100%). Needle is inserted directly into column or insert
Used for dilute samples (< 0.1%) directly above the column.
Injection speed slow. Widely used for :
Should not be performed isothermally. o Trace analysis
Solvent focusing is important. o Thermally labile compounds e.g Pesticides, Drugs
Controlled by solenoid valves. o Wide boiling point range
Requires careful optimisation. o High molecular weight compounds


2. Injection system
Samples Injection type : Large Volume Injection
To enhance sensitivity in Environmental applications.
Uses 100L syringe: Inject up to 70 L.
Very slow injection with injector temperature of a few
degrees below solvent boiling point.
Solvent vents out of split vent, thus concentrating the
Fast temperature ramp to top column temperature
Column programming as per sample requirements