Chromatography (PGE 09)
By: Merna Maged
[Link]
It is a physical method used
mainly for the separation of
Chromatography
the components of a mixture,
in which the components are
distributed between two
phases, one of which is
stationary and the other is
mobile phase.
ANALYSE IDENTIFY QUANTIFY PURIFY
Stationary Phase
Substance that is Liquid or gas
fixed in place, to phase that
which the carries the
Mobile Phase
solvents and sample through
analyte travel the instrument.
through or bind
to it.
• Adsorbent = Stationary Phase
• Adsorptive = Sample
• Solvent = Mobile Phase
Key Words…
• Effluent = any liquid that passes out of
the column (pure or with compound)
• Eluate = Part of effluent that contains
the sample
Eluotropic Series
A series of solvents (eluents) with
an increasing degree of solvent
strength generally used in liquid–
solid or adsorption
chromatography.
In an order of increasing Polarity:
Hexane – Chloroform - Ethyl
acetate – Methanol – Water
A. Separation Procedure
Classification of
Chromatography B. Development Procedure
according to:
C. Method of Packing
A. Separation Procedure
Ion
Adsorption Partition Exchange
Size
Exclusion
Affinity
Adsorption
Chromatography
It depends on intermolecular
forces between solute molecules
and atoms of solid stationary
phase, as: Vander Wal forces,
electrostatic forces and hydrogen
bonds.
What is the
difference
between
Adsorption
and
Absorption?
Normal Phase vs Reversed Phase Adsorption
Chromatography
P.O.C Normal Phase Reversed Phase
Stationary Phase Polar (e.g.: Silica or Alumina) Non-polar (e.g.: long chain
C18 reversed silica)
Mobile Phase Non-polar Polar
Order of Elution Non-polar compounds are Polar compounds are eluted
eluted first first
Note
Functional
groups in
COOH > OH >
order of
NH2 > C=O
decreasing
polarity:
Question???
• It depends on the distribution of a
mixture of components between
two immiscible liquids and
depends on the portioning of the
co-efficient.
Examples:
Partition Paper Chromatography and
Chromatography Droplet Counter Current
• Partition Co-efficient = Conc. of
components in stationary phase /
Conc. of components in mobile
phase.
It depends on the molecular
weight and size of the
components where the larger
molecules are eluted first.
Size Exclusion Sephadex gel is the most
Chromatography common stationary phase used.
Gel Electrophoresis is an
application on Size Exclusion
Chromatography.
Ion Exchange
Chromatography
Ion exchange chromatography is a
process that allows the separation of
ions and polar molecules based on
their affinity for the ion exchanger. It
can be used for almost any kind of
charged molecule, including large
proteins, small nucleotides, and amino
acids
It is highly used in the analysis of amino
acids.
To determine the base composition of
nucleic acids. Chargaff used this technique
to establish Adenine and Thymine’s
Applications on equivalence; Guanine and Cytosine.
Ion Exchange
Chromatography This is the most effective method for
water purification.
Protein purification
It is also used to separate many vitamins,
other biological amines, and organic acids
and bases.
Affinity
Chromatography
It is a separation method
based on a specific binding
interaction between an
immobilized ligand and its
binding partner.
Example: Enzyme with an
inhibitor, antigen with an
antibody.
Principle of Affinity Chromatography
The stationary phase consists of a support medium, on which ligand is
bound covalently, in such a way that the reactive groups that are
essential for binding of the target molecule are exposed.
As the crude mixture of the substances is passed through the
chromatography column, substances with binding site for the immobilized
ligand bind to the stationary phase, while all other substances are eluted.
Once the other substances are eluted, the bound target molecules can
be eluted by methods such as including a competing ligand in the mobile
phase or changing the pH, ionic strength or polarity conditions.
Its applications include:
• Separation of mixture of
compounds.
• Removal of impurities or in
purification process.
• In enzyme assays
• Detection of substrates
• Investigation of binding sites of
enzymes
• In in vitro antigen-antibody
reactions
B. Development Development
Procedure
Elution
Displacement
Frontal Analysis
Development
The mobile phase flow is stopped before solutes reach the
end of the bed of stationary phase. The mobile phase is called
the developer, and the movement of the liquid along the bed
is referred to as development.
Developers are stronger than solvents but are less adsorbed
than adsorptive (sample). They cause moving of zones in the
chromatogram by competing favourably with adsorptive for
the stationary phase.
Elution
The most commonly used chromatographic method in which a
sample is applied to the head of the column and individual
analytes are separated and eluted from the end of the
column.
Eluents have more eluting power than developers and serve to
remove the adsorptive outside the column.
Elution
The are two techniques: Isocratic Elution and Gradient elution.
Isocratic Elution Gradient Elution
the mobile-phase composition is held the mobile-phase composition will be varied
constant during separation of the sample during sample separation
e.g. 60% v/v acetonitrile–water e.g. changing from 0% to 100% v/v
acetonitrile–water
Displacement
Displacers are liquids that are more strongly
adsorbed than adsorptive, causing its
displacement and expelling from the adsorbent to
the mobile phase.
Frontal
Analysis
A form of chromatography where the sample is
continuously added to the column.
Used in Affinity Chromatography and Droplet Counter
Current.
Tailing
The problem of tailing in PC and TLC Tailing causes overlapping of spots.
This may be due to the increased concentration of the sample to be
chromatographed or using unsuitable developing solvent system.
This problem can be solved by:
1. Dilution of the sample.
2. Changing the mobile phase (using different polarity of the solvent system).
3. Changing the pH of the mobile phase by adding either acid or base
depending on the nature of compounds.