CCHAPTER 7C BY JAMIL MOHAMED SAPARI

BASIC CHROMATOGRAPHY
- Column Chromatography

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 ADVANTAGES APPLICATION
2 COLUMN
CHROMATOGRAPHY
VERSUS OF
DISADVANTAGES CHROMATOGRAPHY
2. COLUMN CHROMATOGRAPHY

A method used to purify individual

chemical compounds from a mixture
of compounds.

The stationary phase is held in a

narrow tube and the mobile phase is
forced through the tube by gravity.

Use a filter (a glass frit or glass wool

plug) to prevent the loss of the
stationary phase at the bottom.

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 Column Chromatography
Column or elution The individual components are
retained by the stationary phase
chromatography is a type of
adsorption chromatography.
1 4 differently and separate from each
other while they are running at
different speeds through the column
with the eluent.
Stationary phase is finely divided
inert solids such as alumina (Al2O3), At the end of the column they elute
silica or cellulose powder placed in a 2 5 one at a time. During the entire
narrow tube or column. chromatography process the eluent
is collected in a series of fractions.

Mobile phase is a pure solvent or mixture of The composition of the eluent flow can be
different solvents such as mixture of hexane
and ethyl acetate.
3 6 monitored and each fraction is analyzed
for dissolved compounds e.g. by analytical
chromatography, UV absorption, or
fluorescence.
IDEA
 CONTINUE…. • Elution is a process in which solutes are
washed through a stationary phase by the
movement of a mobile phase.

• An eluent is a solvent used to carry the

Loaded
sample
components of a mixture through a stationary
phase.
Stronger
interaction

• Faster flow rates can be achieved by using a

pump or compressed gas (air, nitrogen,
Moderate
interaction argon) to push the solvent through the
Weaker
interaction
column.

• Two methods are generally used to

prepare a column:
i. Dry method
ii. Wet method 5
DRY METHOD WET METHOD

Mobile phase
Column is filled with
Column is filled with slurry of stationary
dry stationary phase phase powder and
Sand
eluent.
powder
This layer is usually

Stationary phase
Followed by the Solution of organic topped with a small
addition of mobile material is pipetted on layer of sand or with
top of the stationary
phase, phase.
cotton or glass wool to
protect the shape of the
organic layer from the
Then flushed through Sand velocity of newly added
the column until it is Glass wool
Eluent is slowly eluent.
completely wet. passed through
the column to
DRY METHOD
advance the
organic material

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 Separation of compounds based on polarity
1. Perform a column chromatography using a 2. Perform a column chromatography using a
non-polar (SP) stationary phase and a polar (SP) stationary phase and a non-polar
polar (MP) mobile phase. (MP) mobile phase.

– Compounds will be separated due to different – Compounds will be separated due to different
solubility in mobile phase and stationary phase. solubility in mobile phase and stationary phase.

– The more polar compound is more soluble in – The less polar compound is more soluble in the
the mobile phase. So, it will be eluted faster. mobile phase. So, it will be eluted faster.

– The less polar compound is more attracted to – The more polar compound is more attracted to
the stationary phase. It will retained stronger to the stationary phase. It will retained stronger to
the stationary phase and will be eluted slower. the stationary phase and will be eluted slower.

– Polar compound will eluted first followed by the – Less polar compound will eluted first followed
less polar compound. by the polar compound.

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ADVANTAGES DISADVANTAGES

Low cost and disposability of the

stationary phase. Requires some technical skill and
manual dexterity.

Determine the number of

components of a mixture.
Take time to prepare and use.
Separate and purify substantial
quantities of those components for
subsequent analysis.

Requires constant attention while the

Can be scaled to the project at hand. experiment is being performed.

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 Comparison between CC, PC and TLC

COLUMN CHROMATOGRAPHY PAPER CHROMATOGRAPHY THIN LAYER CHROMATOGRAPHY

Water trapped in the paper or

paper strip which A solid adsorbent such as
Stationary phase is placed
STATIONARY PHASE made up of cellulose fibers alumina (Al2O3) or
in a narrow tube or column.
(made up of hydroxyl silica gel (SiO2).
Groups -OH).

MOBILE PHASE Liquid Liquid Liquid

TYPE OF
Adsorption Partition Adsorption
CHROMATOGRAPHY
TYPE OF
Planar chromatography
CHROMATOGRAPHY Column chromatography Planar chromatography

TYPE OF FORCE THAT

MOVES THE MOBILE Gravity Capillary Action Capillary Action
PHASE 9
Silica gel for open column chromatography
mobile phase: solvent (liquid)

stationary phase: silica gel (solid)

Thin-layer chromatography (TLC)

stationary phase: TLC plate (a solid supported
on an inert plate)

mobile phase: solvent (liquid)

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 APPLICATIONS OF
PLANAR AND COLUMN
CHROMATOGRAPHY

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 APPLICATIONS OF CHROMATOGRAPHY
➢ Pharmaceutical Company
- determine amount of chemical in new product

➢ Hospital
- detect blood or alcohol levels

➢ Law Enforcement
- compare a sample found at a crime scene with samples
from suspects

➢ Environmental Agency
- determine the level of pollutants in the water supply

➢ Manufacturing Plant
- to purify a chemical needed to make a product
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Applications of TLC

• Analyzing ceramides and fatty acids.

• Detection of pesticides or insecticides in food and water.

• Analysing the dye composition of fibers.

• Identification of medicinal plants and their constituents.

• In food industry, TLC method is used for separation and

identification of colours, preservatives and sweetening agent.
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 Applications of Planar Chromatography

To separate the coloured To separate the components in To separate the components

pigments in plants. a mixture, for example a of inks and dyes, plant
mixture of amino acids. compound, make-up and many
other substances. 14
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