CHROMATOGRAPHY
ENVE 202
Dr. Aslıhan Kerç
Chromatography is a method
of physically separating
mixtures of gases, liquids, or
dissolved substances.
Chromatography can be used to
identify drugs, poisons and many
other substances.
Separation is determined by the
molecular size and/or charge
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What is chromatography?
Chromatography (from Greek word for chromos for
colour) is the collective term for a family of laboratory
techniques for the separation of mixtures. It involves
passing a mixture which contains the analyte through a
stationary phase, which separates it from other molecules
in the mixture and allows it to be isolated.
Which means ...
Chromatography is the physical separation of a mixture
into its individual components.
http://members.shaw.ca/vict/chemistry_test3.htm
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 All forms of chromatography involve 2 phases
 The Two Phases
 1. stationary (absorbent) phase
o the material on which the separation takes place
 2. mobile phase - the solvent transports the sample
 Types of Chromatography - 5 of them
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Separation
A sample is often a mixture of many components in a
complex matrix.
The components must be separated from each other so that
each individual component can be identified by other
analytical methods.
The separation properties of the components in a mixture
are constant under constant conditions.
Once determined they can be used to identify and quantify
each of the components.
A mixture can be separated using differences in physical
or chemical properties of the individual components.
In chromatography
A column holds the stationary phase, and the mobile
phase carries the sample through it.
Sample components that partition strongly into the
stationary phase spend a greater amount of time in the
column and are separated from components that stay
predominantly in the mobile phase and pass through the
column faster.
Chromatograms
If a detector that responds to solute concentration is placed
at the end of the column and its signal is plotted as a
function of time, a series of symmetric peaks is obtained.
The positions of the peaks on the time axis is used to
identify the components of the sample.
The areas under the peaks provide a quantitative measure
of the amount of each species.
Gas Chromatography
-
Gas Chromatography
a gas separates the components
they are then ionized and an electrical signal is
recorded
mobile phase - carrier gas
stationary phase
- thin film of liquid
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HPLC
 High Performance Liquid Chromatography (HPLC)
 - done at room temp. - advantageous for chemicals
that are heat sensitive or volatile - example LSD
 - a liquid (mobile phase) is pumped through a
column containing fine particles (stationary phase)
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TLC
 Thin - Layer Chromatography (TLC)
 -A plate is coated with a granular gel - usually
silica gel or aluminum oxide (stationary phase)
 - the substance to be separated is carried up the
plate by capillary action - the substance with most
affinity for the plate will rise the farthest.
 4. Paper chromatography
• Same as TLC but paper
• is used as stationary phase
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Electrophoresis
Electrophoresis
- Similar to TLC but the substance is separated
through a gel by electric current
- Due to different size and charge substances will
move across the plase at different speeds.
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Paper Chromatography of Ink
Two samples of black
ink from two different
manufacturers have
been characterized
using paper
chromatography.
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Retention Factor (Rf)
 A number that
represents how far a
compound travels in a
particular solvent
 It is determined by
measuring the distance
the compound traveled
and dividing it by the
distance the solvent
traveled.
Solve for Rf
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