Clinical Analytical Chemistry
CLS 231
Introduction to Instrumental
Analysis
- Chromatography
Lecture 12
Lecturer: Amal Abu-Mostafa
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Session Objectives:
What is chromatography?
The principle of chromatography.
Chromatography is used in.
Classification of Chromatographic Methods.
Column chromatography.
Classification of column chromatography.
Planar chromatography.
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What is chromatography?
Chromatography (from Greek word chromos for colour) is
the collective term for a family of laboratory techniques for the
separation of mixtures.
It is a technique used to separate and identify the components of a
mixture.
Mobile phase
Stationary phase
Column
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The principle of chromatography
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.
During the process of chromatography, the components of a
sample distribute themselves between the mobile phase
(which may be a gas, a liquid, or a supercritical fluid.) and
stationary phase (, which is fixed in place in a column or on a
solid surface).
Molecules that spend most of their time in the mobile phase are
carried along faster.
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Chromatography is used in:
• Chromatography is a widely used method that allows the
separation, identification, and determination of the chemical
components in complex mixtures (such as proteins, vitamins, sub
cellular compartments…. etc.).
• We can use chromatography to separate the components of inks
and dyes, such as those found in pens, markers, clothing, and even
candy shells.
• Chromatography can also be used to separate the colored pigments
in plants or used to determine the chemical composition of many
substances.
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Classification of Chromatographic Methods
Chromatographic methods can be
categorized in two ways.
The first according to the shape of the
stationary phase:
1) Column chromatography
2) Planar chromatography
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Classification of Chromatographic Methods
1) Column chromatography
In column chromatography, the stationary phase is
held in a narrow tube through which the mobile
phase moves under the influence of gravity or
pressure.
Mobile phase
Stationary phase
Column
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General Theory of Column Chromatography
A typical column chromatography experiment is
outlined in Figure 12.4.
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General Theory of Column Chromatography
The sample is introduced at the top of the column as a narrow band.
Ideally, the solute’s initial concentration profile is rectangular (Figure
12.5a).
As the sample moves down the column the solutes begin to separate,
and the individual solute bands begin to broaden and develop a
Gaussian profile (Figures 12.5b,c).
If the strength of each solute’s interaction with the stationary phase is
sufficiently different, then the solutes separate into individual bands
(Figure 12.5d).
The progress of a chromatographic separation is monitored with a
suitable detector situated at the end of the column. A plot of the
detector’s signal as a function of time or volume of eluted mobile
phase is known as a chromatogram (Figure 12.6) and consists of a
peak for each of the separated solute bands.
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A more fundamental classification of Column
chromatographic methods is one based upon
the types of mobile phases:
(1) liquid chromatography
(2) gas chromatography
(3) supercritical-fluid chromatography.
The mobile phases in the three techniques are liquids, gases, and
supercritical fluids respectively.
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Classification of column chromatography
Liquid chromatography includes the following
methods:
(a) Ion-exchange chromatography
(b) Size-exclusion chromatography
(c) Affinity chromatography
(d) HPLC, or high-performance liquid chromatography
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For example three of the column chromatographic methods
are used in protein purification (a), (b) and (c).
(a) Ion-exchange chromatography
Ion-exchange chromatography A form of liquid
chromatography in which the stationary phase is an
ion- exchange resin.
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(a) Ion-exchange chromatography
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(a) Ion-exchange chromatography
In ion-exchange chromatography (IEC) the stationary
phase is a cross-linked polymer resin, usually
divinylbenzene cross-linked polystyrene, with
covalently attached ionic functional groups.
The counterions to these fixed charges are mobile and
can be displaced by ions that compete more favorably
for the exchange sites.
Ion-exchange resins are divided into four categories:
strong acid cation exchangers; weak acid cation
exchangers; strong base anion exchangers; and weak
base anion exchangers.
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(b) Size-exclusion chromatography
Size-exclusion chromatography: A form of liquid
chromatography in which the stationary phase is a
porous material and in which separations are based on
the size of the solutes.
In size-exclusion chromatography, also called
molecular-exclusion or gel-permeation
chromatography, separation is based on the solute’s
ability to enter into the pores of the column packing.
Smaller solutes spend proportionally more time within
the pores and, consequently, take longer to elute from
the column.
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(b) Size-exclusion chromatography
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(c) Affinity chromatography
The stationary phase is a group specific liquid bonded to a solid
surface.
Affinity chromatography Separates proteins by their binding
specificities.
The proteins retained on the column are those that bind
specifically to a ligand cross-linked to the beads.
(In biochemistry, the term “ligand” is used to refer to a group
or molecule that binds to a macromolecule such as a protein.)
After proteins that do not bind to the ligand are washed
through the column, the bound protein of particular interest is
eluted (washed out of the column) by a solution containing
free ligand.
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(c) Affinity chromatography
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(d) High-performance liquid chromatography HPLC:
High-performance liquid chromatography: A
chromatographic technique in which the mobile
phase is a liquid also.
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Classification of Chromatographic Methods
2) Planar chromatography
In planar chromatography, the
stationary phase is supported on a flat
plate or in the interstices of a paper;
here, the mobile phase moves through
the stationary phase by capillary
action or under the influence of
gravity.
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Example on Planner Chromatography
Thin-Layer
Chromatography
Uses thin plastic or glass
trays
to
identify
the
composition of pigments,
chemicals,
and
other
unknown substances.
Paper Chromatography
Can be used to separate the components of inks, dyes, plant
compounds (chlorophyll), make-up, and many other
substances
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Thank you
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