CHM 256 7a

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CHAPTER 7

BASIC
CHROMATOGRAPHY

JAMIL MOHAMED SAPARI

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Learning Outcomes
At the end of the course, student should be able to explain:

Principle of chromatography

Types of chromatography - on the basis of interaction


of the analyte with stationary phase

Types of chromatography and techniques


- Planar chromatography and column chromatography

Applications of planar and column chromatography

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PRINCIPLE OF CHROMATOGRAPHY

1 Samples to be analysed are usually a mixture of many components in a complex


matrix.

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For samples containing unknown compounds, the components must be
separated from each other so that each individual component can be identified
by other analytical methods.

3 Measurement techniques used for chemical analysis are usually specific for a
single chemical species.

4 A mixture can be separated using the differences in physical or chemical


properties of the individual components.

5 Example of physical properties that are useful for separations are density and
size.

6 Some useful chemical properties by which compounds can be separated are


solubility, boiling point, and vapor pressure.
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CLASSICAL SEPARATION METHODS


• Masking
• Precipitation
• Distillation
• Solvent extraction
• Ion exchange
• Chromatography
• Electrophoresis
CHROMATOGRAPHY Illustration of Chromatography

✓Chromatography is a technique used to separate the


components of a mixture based on the rates at which they are
carried through a stationary phase by a gaseous or liquid phase
known as the mobile phase.

✓ All types of chromatography contain a stationary phase (solid or liquid) to adsorb or absorb the mixture being
separated and a mobile phase (liquid or gas) which passes over the stationary phase and competes with it for the
constituents of the mixture.

✓ The two phases are:


1) the stationary phase
2) the mobile phase
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USES OF CHROMATOGRAPHY
Chromatography is used by scientists to:

Analyze Identify Purify Quantify


• examine a • determine the • separate • determine the
mixture, its identity of a components in amount of the a
components, and mixture or order to isolate mixture and/or
their relations to components based one of interest the components
one another. on known for further study. present in the
components sample.

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PRINCIPLE OF CHROMATOGRAPHY

Competition
Sample is
between the
dissolved in a
sample molecules
solvent and
adsorbed by the
washed through a
stationary phase
stationary phase
and dissolved by
by a mobile phase
the eluent results
called eluent. Components in the mixture separate out
in separation.
as the mobile phase moves through
Substances that
Speed of the
the stationary phase.
are more soluble
substance passes
in the eluent and
through depends
less adsorbed by
on the nature of
the stationary
the stationary
phase move faster
phase and the
through the
eluent.
apparatus.

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Principles of chromatographic separations
Sample Mobile phase

Component A
Stationary
Component B
phase
Component C

• A small volume of sample is placed at the top of the column (filled with stationary phase and
solvent).
• Sample is eluted with mobile phase.
• The individual components interact with the stationary and mobile phase to different degrees.
• Component A interact more strongly to the stationary phase than component B and component C.
• Then, as the mixture travels down the column, component A will be retarded with respect to the
component B and C.
• In time, they will separate into bands, and be eluted at different times.
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• Retention time
Time taken for a component in the sample to pass through the column.

• Chromatogram
Visual output of the chromatograph. Different peaks or patterns on the
chromatogram correspond to different components of the separated mixture.

X-axis - retention time


Y-axis – signal corresponding to the
response created by the analytes existing
in the system
Types of chromatography
- on the basis of interaction of the
analyte with stationary phase

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Types of chromatography on the basis of interaction
of the analyte with stationary phase

Adsorption Partition

Ion exchange Size exclusion

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In the adsorption process, 1. ADSORPTION
CHROMATOGRAPHY
the solute molecules are
The separation of solute
held on or attached on
depends on the different
the surface of the
in polarity.
stationary phase which is
solid. o Definition: A
chromatography where
the analytes
(components) are
separated based on their
Because of the different different degree of
degree of intermolecular
attraction, some adsorption (stick) on a
components of the surface of solid
mixture will be more stationary phase.
attracted or adsorbed to
the stationary phase thus
separated.

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1

o Substances that
are strongly 3
adsorbed move
through the o The stationary
stationary phase phase is a polar
much more slowly solid, usually finely 4
than those that are divided silica and
weakly adsorbed. alumina and the oThe separation of
solutes are polar the solutes depends
molecules. on the difference in
their polarity. The
more polar solutes oMobile phase may
are more readily be a liquid (LSC) or a
adsorbed than the gas (GSC).
less polar solutes.
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ADSORPTION
CHROMATOGRAPHY

Stationary
phase: solid

Mobile
phase: liquid

solute

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o Example:
1. Thin-layer chromatography (TLC)
- stationary phase: plane (a solid supported on
an inert plate)
- mobile phase: liquid (solvent)

2. Column chromatography
- Stationary phase: silica gel (solid)
- Mobile phase: solvent (liquid)

3. Gas solid chromatography


- Stationary phase: solid
- Mobile phase: gas

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2) PARTITION CHROMATOGRAPHY

❖Partitioning is the distribution (by dissolving) of the components between 2


immiscible phases. Separation of the components will be based on relative
solubility of the componentsin the mobile phase and stationary phase

❖Also known as liquid- liquid chromatography.

❖Stationary phase is a liquid supported on an inert solid (surface of packing


particles).
❖Mobile phase may be a liquid (LLC) or gas (GLC).

❖Separation is based on solute partitioning between two liquid phase (relative


solubility).
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PARTITION CHROMATOGRAPHY

Mobile phase:
liquid

Stationary solute
phase: liquid
supported on
solid
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❖ Examples:
i. High performance liquid chromatography (HPLC)
ii. Paper chromatography
iii. Gas chromatography (GC)

HPLC GC Paper chromatography


❖ Two main types of partition chromatography based on the type of stationary phase:
• normal-phase liquid chromatography
• reversed-phase liquid chromatography

Normal-phase chromatography Reversed-phase chromatography


• Stationary phase: polar stationary phase • Stationary phase: nonpolar stationary
(e.g., methanol on silica gel). phase
• Mobile phase: nonpolar mobile phase • Mobile phase: polar mobile phase.
(e.g., hexane). • Separation: Nonpolar solutes are retained
• Separation: Favours retention of polar more and polar solutes more readily
compounds and elution of nonpolar eluted.
compounds.

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TYPES OF CHROMATOGRAPHY AND TECHNIQUES

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PLANAR CHROMATOGRAPHY
COLUMN CHROMATOGRAPHY (CC)
TYPES OF CHROMATOGRAPHY

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COLUMN AND PLANAR CHROMATOGRAPHY

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TRY THIS !!!

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