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Introduction to Instrumental Analysis - Chromatography
Clinical Analytical Chemistry CLS 231 Introduction to Instrumental
Analysis - Chromatography Lecture 12 Lecturer: Amal Abu-Mostafa
What is chromatography? The principle of chromatography.
Session Objectives: What is chromatography? The principle of
chromatography. Chromatography is used in. Classification of
Chromatographic Methods. Column chromatography. Classification
ofcolumn chromatography. Planar chromatography. What is
chromatography?
Chromatography (from Greek wordchromosfor 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 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 alongfaster.
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. 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 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 General Theory of Column Chromatography
A typical column chromatography experiment is outlined in Figure
12.4. General Theory of Column Chromatography
The sample is introduced at the top of the column as a narrow band.
Ideally, the solutes 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 solutes 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 detectors 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. 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.
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 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. (a) Ion-exchange chromatography (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. (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 solutes 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. (b) Size-exclusion chromatography
(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 bindspecifically to a ligand cross-linked to the beads.
(In biochemistry, theterm ligand is used to refer to a group or
molecule that binds to amacromolecule such as a protein.) After
proteins that do not bind tothe ligand are washed through the
column, the bound protein ofparticular interest is eluted (washed
out of the column) by a solutioncontaining free ligand. (c)
Affinity chromatography (d) High-performance liquid chromatography
HPLC:
High-performance liquid chromatography: A chromatographic technique
in which the mobile phase is a liquid also. 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. Example on
Planner Chromatography
Paper Chromatography Can be used to separate the components of
inks, dyes, plant compounds (chlorophyll), make-up, and many other
substances Thin-Layer Chromatography Uses thin plastic or glass
trays to identify the composition of pigments, chemicals, and other
unknown substances. Thank you