Download Chromatography (Principles and Classifications)

Chapter 4-1
Chromatography
Is a technique used to separate
and identify the components of
a mixture.
Dr Gihan Gawish
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 Before a protein or other biological macromolecule can be rigorously
studied from a structural and functional basis, it must be purified.
 The problems that can arise during protein purification become clear
when one considers that a single protein has to be purified from a
mixture of as many 10,000 proteins, each of which are made up of
the same constituent amino acids.
 Proteins differ in size (how many amino acids), charge (how many
positively and negatively charged amino acids), and in sequence and
presence of specific binding sites on the proteins.
 Any technique that could be used to purify protein must be based on
these inherent differences.
Dr Gihan Gawish
Chromatography Application in Biomolecules
General Principles of Chromatography
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
Separation of molecules by distribution between a
stationary phase and a mobile phase.
A
stationary phase can be solid, gel, or liquid. Also called
matrix, resin, or beads.
 The
mobile phase is the solvent and it is usually a liquid, but
may also be a gas. Also called eluting buffer
 The
compounds to be separated are considered
solutes
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Basis of Chromatography
Differential retardation of materials caused by
interactions with the stationary phase:
Reservoir
+ Sample
mobile phase
stationary phase
or Resin
Frit
Flow-through
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Classification of Chromatography

by mobile phase:
1.
2.

by stationary phase or shape
1.
2.
3.
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Gas chromatography,
Liquid chromatography or its variations
Paper chromatography,
Thin-layer chromatography
Column chromatography
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Classification by the Separation mode
The mechanism that causes the stationary phase to
retard the movement of molecules
1.
Sieve mechanism  separation according to size or
MW.
(molecular sieve = gel filtration = size exclusion = gel
permeation)
2.
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Charge interaction  separation based on net charge
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Classification by the Separation mode
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3. Solubility characteristics  separation based on
polarity
Hydrophobic chromatography, reverse-phase chromatography,
Adsorption or normal-phase chromatography
4. Biological or Specific interaction  capture any molecule
that exhibit such property

affinity chromatography, dye-chromatography

Antibody-antigen: (Immuno precipitations and other forms)
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Gel Filtration Chromatography
also called "Molecular
Sieve“ or "Size Exclusion“:
(column
matrix) "beads" of
hydrated,
porous
polymer.

Stationary phase

Mobile phase:
solvent.
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buffer or
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Gel Filtration Chromatography
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Concept of separation: molecules "filter" through the
porous beads:
1.
2.
3.
Large molecules can’t get through any pores in the
beads and move more rapidly through the column,
emerging (eluting) sooner.
Smaller molecules may enter some pores in the
beads, and thus, have more space to "explore" 
elute later.
Ions and small molecules can enter Drthrough
all pores
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of the beads  elute last
Gel Filtration Chromatography
The porous beads represent
a restricted space or volume

Molecules may diffuse to
this space if permitted by
pore size
Absorption

Vo
10
Ve
Volume
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Vt
Gel Filtration Chromatography
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For every column, three volumes should be distinguished:
1. The void volume, Vo: which is the volume external to the beads
 This space is accessible to all molecules entering the gel
 Can be determined by the elution of HMW molecules
2. The total volume, Vt:
 can be calculated from the dimension of the column
3. The elution volume, Ve, of molecules
 All molecules must eluted between the void volume and total
volume of the column
Dr Gihan Gawish
Gel Filtration ChromatographyPartition Coefficient

In classical and more rigorous science, elution position
of any molecules should be reported as partition
coefficient (Kav) rather than volume.
Kav=
12
(Ve-Vo)
(Vt-Vo)
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Gel Filtration Chromatography
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


Kav represents the fraction of bead-restricted
volume into which a solute could partition
Kav ranges between 0 and 1 for all molecules
Kav allows comparison between columns of
different dimensions and/or materials
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Examples of Partition Chromatography
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With adsorptive effect
1- Paper.
3- Gel
Without adsorptive effect
2- Thin layer
4- Gas liquid
Column
Support: sillica gel, cellulose powder
or dextran (washed or suspended of
solvent)
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Adsorptive Chromatography (adsorptive effect)
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A
stationary phase solid (support; matrix) + liquid
(adsorbent)
 The

mobile phase is the solvent
The relative movement of each molecule is a result
of a balance between the driving force and
retardation force.
Dr Gihan Gawish
Common Materials Used in Adsorption
Chromatography in Biochemistry
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Material
Alumina
Silica gel
Activated carbon
Calcium phosphate gel
Hydroxyapatite
Substance separated
Small organic molecules,
proteins
Sterols, amino acids
Peptides, amino acids,
carbohydrates
Proteins, polynucleotide
Nucleic acids
Dr Gihan Gawish