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Chromatography
By Dr Ashish Agravatt
Chromatography
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Introduction of Chromatography
Definition
Chromatography is a separation technique based on the different interactions/diiferential distribution of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium.
Components:
mobile phase: a solvent that flows through the supporting medium
stationary phase: a layer or coating on the supporting medium that interacts with the analytes
supporting media: a solid surface on which
the stationary phase is bound or coated
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Types of Chromatography…
Paper
HPLC
Gas
Thin layer
Column
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Types of chromatography
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Paper chromatography
⮫ Stationary phase:
Paper Whatman paper 1 or 3 DEAE Cellulose (Diethylaminoethyl) CM Cellulose (Carboxy methyl)
⮫ Mobile phase: Mixture of organic solvants, water & additatives.
⮫ Ascending & descending chromatography.
⮫ Amino acids, sugars and some drugs or chemicals separated.
⮫ Diagnosis of Aminoacidurias (Phenylketouria, Alkaptonuria etc.
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Paper
Strip
Mobile
Phase
Mobile
Phase
PAPER CHROMATOGRAPHY
ASCENDING
DESCENDING
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DETECTION
"Rf value is the ratio of distance travelled by
the solute to the distance
travelled by the solvent front”
Distance travelled by solute
Rf = Distance travelled by solvent front
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THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
The Rf is defined as the distance the center of the spot moved divided by the distance the solvent front moved (both measured from the origin)
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Thin layer chromatography
⮫ Adsorption TLC
⮫ Stationary phase: Silica gel, Alumina
water & additatives.
⮫ Differential adsorption & partition coefficient of components between stationary & mobile phase.
⮫ Eg. Phospholipids and pigments.
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COLUMN CHROMATOGRAPHY
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Column Chromatography
Stationary phase is held in a narrow tube through which the mobile phase is forced under pressure or under the effect of gravity
Column chromatography is generally used as a purification technique
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MOBILE PHASE
STATIONARY PHASE
ELUTE
COLUMN CHROMATOGRAPHY
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Columnar Chromatography (CC)
The stationary phase is packed into a column.
The mobile phase is a moving liquid or gas.
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Liquid chromatography
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SEPARATION MECHANISMS
1- Adsorption chromatography.
2- Partition chromatography.
3- Ion exchange chromatography.
4- Gel filtration chromatography.
5- Affinity chromatography
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� Principle of separation�
Chromatography in which separation is based mainly on differences between the adsorption affinities of the sample components for the surface of an active solid.
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Ion-exchange Chromatography
Separation is based on an exchange of ions between a charge stationary surface and mobile phase of opposite charge. (Between surface and eluents).
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Operationally, the particle surfaces of a plastic resin or silica serve as the stationary phase to which funtional groups with fixed cationic or anionic charges are bounded.
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Cation-exchange particle- contains covalently
bound negatively charge functional groups.
* Strong acidic groups- sulphonate ions.
* weak acidic groups – carboxylate ions
- phosphate(p)
- sulfomethyl
- sulfoethyl
- sulfopropyl
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Anion- exchange packing –
they are strongly basic quaternarly
amines with positive charges.
Used to separate anionic solutes.
Examples- triethylaminoethyl groups
or
Weakly basic groups – such as
* aminoethyl(AE)
* diethylaminoethyl (DEAE)
* guanidoethyl(GE).
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Clinical application:-
-Separation of aminoacids, peptides,
proteins , nucleotides, oligonucleotides
and nucleic acid.
- Separation and removal of inorganic ions from aqueous mixtures.
*Most water purification units used to prepared deionized water for the laboratory contains “ mixed-bed” columns of cations and anion resins.
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Gel-Filteration Chromatography
Also known as
* Size-Exclusion Chromatography
* Gel- Permeation Chromatography
* Molecular seive Chromatography
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Separation of the basis of their
molecular size.other factors are
molecular shape & hydration.
- Beads of these materials are porous with
pore size that allow small molecules to
be temporarily entrapped.
-Too larger molecules do not enter pores
remain entirely in the mobile phase
and are rapidly eluted from columns.
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Stationary phase :- include
* cross-linked dextran
* agarose
* porous glass
* polyacrylamide
* polystryrene-divinylbenzene
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Applications of GPC to natural products
Determination of M. wt. of peptides, proteins & polysaccharides.
Separation of mixture of mono- & polysaccharides.
Separation of amino acids from peptides & proteins.
Separation of proteins of different molecular weights.
Separation of myoglobin & haemoglobin.
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Affinity Chromatography
Separtion is based unique and specific biological interaction of the analytes and ligand is used for the separtion.
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Affinity chromatography
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Clinical application:-
*To separate and prepare larger
quantities of protein and antibodies
for futher study.
*Cells with different suface carbohydrate moietes are seperated by lectin columns.
*Low-density and very low density lipoprotein are separated with heparin columns.
*Glycated hemoglobins are separated with phenylboronate columns.
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Gas Chromatography
Developed by James and martin
in 1952 to separate fatty acids.
Gaseous mobile phase is used to pass a mixture of volatile solutes through a column containing stationary phase.
Here mobile phase is a typically inert gas such as – nitrogen, helium, hydrogen or agron .
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differences in the solutes vapour pressures
and interactions with the stationary phase.
before a less volatile one.
the dectector in the order of their elution.
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their similar retension times.
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Subclassification
GSC
Seperation occurs primarily by differences in adsorption at the solid phase surface.
GLC
Separation is based on differences in solute partitioning between the gaseous mobile phase and liquid stationary phase.
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HPLC
When the particles of small diameters are
used as the stationary phase support the
technique is known as HPLC
Here column efficiency is inversely related to the column packing particle size and pressure drop is related to the square of the particle diameter, relatively high pressure are required to pump liquids through efficient HPLC columns.
Most widely used form of LC.
.
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High pressure use to pump the mobile phase through
a tightly packed column. (5000 – 10,000psi).
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Detectors are ultraviolet absorptimeter.
⮫ Purified fraction of sample mixture recovered.
⮫ High speed, high resolution and versatility.
⮫ Use estimation and detection of hormone -
eg. Epinephrine, nor-epinephrine, ACTH,
Vitamin-A, Calcitriol
Drugs – Phenytoin, Phenobarbitone.
Metabolites – Metanephrins.
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Instrumentation
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LC column
LC injector
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MCQ 1
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MCQ 2
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