Chromatographic

Techniques

PH 762 Experimental Techniques

Dr. Zulqurnain Ali
Some Types of Separation Techniques

– Filtration
– Mechanical separation
– Floatation
– Centrifugation
– Distillation
– Crystallization
– Chromatography
Introduction

Chromatography is an analytical technique commonly used for separating a mixture of chemical

substances into its individual components, so that the individual components can be thoroughly
analyzed.
There are many types of chromatography e.g., liquid chromatography, gas chromatography, ion-
exchange chromatography, affinity chromatography, but all of these employ the same basic
principles.
Problem
I had two reactants ‘A’ and ‘B’. I let them react with each other, under certain reaction conditions, to form a
product ‘C’. After the reaction was complete, I ended up with a reaction mixture that contained unreacted A,
unreacted B and my desired product C. Now my task was to separate out A, B and C to isolate and analyze
pure product C.
 Technique Stationary phase Mobile Basis of separation Notes
phase
Paper solid (cellulose) liquid polarity of compound spotted directly on a cellulose paper
chromatography molecules
Thin layer solid (silica or polarity of glass is coated with thin layer of silica on which is spotted the
chromatography alumina) liquid molecules compound
(TLC)
Liquid column solid (silica or liquid polarity of glass column is packed with slurry of silica
chromatography alumina) molecules
small molecules get trapped in the pores of the stationary phase,
solid while large molecules flow through the gaps between the beads
Size exclusion (microporous liquid size of molecules and have very small retention times. So larger molecules come out
chromatography beads of silica) first. In this type of chromatography there isn’t any interaction,
physical or chemical, between the analyte and the stationary phase.

Ion-exchange solid (cationic or liquid ionic charge of the molecules possessing the opposite charge as the resin will bind
chromatography anionic resin) molecules tightly to the resin, and molecules having the same charge as the
resin will flow through the column and elute out first.
solid (agarose or
porous glass binding affinity of if the molecule is a substrate for the enzyme, it will bind tightly to
beads on to the analyte the enzyme and the unbound analytes will pass through in the
Affinity which are molecule to the mobile phase, and elute out of the column, leaving the substrate
chromatography immobilized liquid molecule bound to the enzyme, which can then be detached from the
molecules like immobilized on the stationary phase and eluted out of the column with an appropriate
enzymes and stationary phase solvent.
antibodies)
gas (inert
Gas liquid or solid gas like boiling point of the samples
comes
are volatilized and the molecule with lowest boiling point
out of the column first. The molecule with the highest
chromatography support argon or molecules boiling point comes out of the column last.
helium)
Term Definition
Mobile phase or carrier solvent moving through the column

Stationary phase or adsorbent substance that stays fixed inside the column

Eluent fluid entering the column

Eluate fluid exiting the column (that is collected in flasks)
the process of washing out a compound through a column using a suitable
Elution
solvent

Analyte mixture whose individual components have to be separated and analyzed

Sepration of different components

Differential affinities (strength of adhesion) of the various components of the analyte towards the stationary and mobile

phase results in the differential separation of the components.

Affinity, in turn, is dictated by two properties of the molecule: ‘Adsorption’ and ‘Solubility’.

Adsorption as the property of how well a component of the mixture sticks to the stationary phase

Solubility is the property of how well a component of the mixture dissolves in the mobile phase

Higher the adsorption to the stationary phase, the slower the molecule will move through the column.

Higher the solubility in the mobile phase, the faster the molecule will move through the column.
Thin Layer Chromatography
In TLC, a plastic, glass or aluminum sheet is coated with a thin layer of silica gel. The
stationary phase is SiO2 and is very “polar”.
A very small amount of a solution of the substance to be analyzed is applied in a small
spot with a capillary tube, ~1cm from the bottom of the TLC plate

As the mobile phase rises up the TLC plate by capillary action, the components dissolve in
the solvent and move up the TLC plate.
Individual components move up at different rates, depending on intermolecular forces
between the component and the silica gel stationary phase and the component and the
mobile phase.

It is capable of strong dipole-dipole and H-bond donating and accepting interactions with the “analytes” (the components
being analyzed).

More polar analytes interact more strongly with the stationary phase in move very slowly up the TLC plate.

By comparison, the mobile phase is relatively nonpolar and is capable of interacting with analytes by stronger London
forces, as well as by dipole-dipole and H-bonding.

More nonpolar analytes interact less strongly with the polar silica gel and more strongly with the less polar mobile phase
and move higher up the TLC plate.
TLC: Calculation of Rf
2.0 cm
Rf (A) = = 0.40
5.0 cm
Solvent Front

Rf (B) = 3.0 cm = 0.60

5.0 cm
Distance solvent
migrated = 5.0 cm
4.0 cm
Distance A
migrated = 3.0 cm Rf (C) = 0.8 cm = 0.16
5.0 cm

Distance B
migrated = 2.0 cm 3.0 cm Rf (D) = 4.0 cm = 0.80
5.0 cm
Distance C
migrated = 0.8 cm
0.8 cm
Rf (U1) = 3.0 cm = 0.60
Origen
x x x x x 5.0 cm
A B U C D
0.8 cm
Rf (U2) = = 0.16
5.0 cm

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)
Size Exclusion Chromatography
Size Exclusion Chromatography

One late night during 1957, as a young scientist was leaving the lab, he made a mistake. The mistake would be a turning

point for his professor, Jerker Porath, and lead to the remarkable discovery of Sephadex (SEparation PHArmacia DEXtran).

Because the young scientist forgot to turn on the power switch when he left the lab that night, Jerker Porath (Uppsala

University, Sweden) realized that a gel made of crosslinked dextran could be used to separate macromolecules—without

applying electrical current. The molecular separation was based purely on size.

The crosslinked dextran would ultimately become Sephadex, the first chromatography medium, and the technique would

be referred to as gel filtration or size exclusion chromatography. Sephadex was an instant success, enabling new scientific

disciplines and paving the way for the biopharmaceutical industry. Since then, GE Healthcare has developed numerous

products for biomolecular separations based on Sephadex.

High Pressure Liquid Chromatography