2. INTRODUCTION
Chromatography is one of the most useful analytical technique by
which closely related molecules in a mixture of biomolecules could be
separated ,isolated and purified. The separation processes is based on
differential distribution of substances between two immiscible phase
including a mobile phase and stationary phase ,one of which moves
relatively to the other.
Based on the type of interaction between the substances and the
stationary phase, chromatographic techniques can be classified into
three major groups
1.Partition Chromatography
2.Permeation or molecular exclusion chromatography
3.Absortion chromatography
3. Based on the type of Stationary phase used, the partition and adsorption
chromatography are further classified into
Partition chromatography- Paper chromatography and Thin Layer chromatography
Adsorption chromatography-Affinity chromatography(AC)
Ion-exchange chromatography
-Cation exchange chromatography
- Anion exchange chromatography
similarly ,based on the type of mobile phase used, the chromatography could be
classified into three types: 1. Liquid chromatography(LC)
2.Gas chromatography(GC)
3. Gas-Liquid chromatography(GLC)
Paper and thin layer chromatography
In paper chromatography ,the paper (made of cellulose) acts as stationary phase and the
solvent system acts as mobile phase. When the mixture of substances moves on a stationary
phase(paper) along with the mobile phase (the solvent flowing along the paper) , the
separation is brought about by continuous partition between the mobile phase and water held
in the paper, where paper together with water acts as an adsorbent which results in
retardation of substances at certain levels of flow on the paper. Thus the paper
chromatographic separation is achieved as a resultant of propelling (mobile) and retardation
(stationary) forces.
4. Similarly ,in thin layer chromatography silica gel coated plates are being used
instead of paper. Here again the separation is achieved by the result partition
between the mobile phase and an inert stationary phase (silica).This is being
commonly used in several laboratories to separate hydrophobic non-polar soluble
substances such as lipids.
In both these techniques, one should consider certain properties of solvents as
mobile phases which include
1. Solvent should be stable in air and when mixed with small quantities acid or
alkaline vapor.
2. Components of the mobile phase should be relatively non-volatile or their
volatilability should be similar to the closed apparatus.
3. The solvent should be removed rapidly and completely from the stationary
phase after the chromatogram has been completed.
4. The solvent should remain homogenous throughout the range of temperature
experienced in laboratory.
5. The solvent should not react with any of the substances to be separated or with
stationary phase.
Identification of compounds separated
An important characteristics used in both paper and thin layer chromatography for
identification of compounds of substances that are separated is identified after using certain
5. This value (Rf) can be calculated by measuring the distances travelled by the
substance(separate) and the solvent( mobile) from their point of origin.
Permeation of Molecular exclusion or Gel filtration chromatography
Gel filtration chromatography is one of the most commonly employed tools for separating
biomolecules on the basis of their molecular weight. Thence it is other wise called as
molecular exclusion or molecular sieve chromatography . This system involves a stationary
phase (usually a preswollen gel beads) and a mobiles phase(buffer system).
Principle: In this technique, the mixture of biomolecules dissolved in a suitable buffer is
allowed to flow by gravity down a column packed with beads of an inert highly hydrated
polymeric material that has previously been washed and equilibrated with the buffer.
During this process , biomolecules of different molecular size penetrate into the internal
pores of the beads to different degrees and thus travel down the column at different rates.
Very large biomolecules cannot enter the pores of the beads,hence excluded and remain in
the exclusion volume of the column. On the otherhand ,small biomolecules which can enter
the gel pores, move more slowly through the column , sinces they spend a proportation of
the their time in stationary phase. Biomolecules of intermediate size will be excluded from
6. the beads to a degree that depends on their size. The biomolecules are therefore
eluted in a order of decreasing molecular size and thus a separation of molecules is
achieved.
The column materials that are commonly used include:
1.Sephadex G-type(G-10,G-25,G-50,G-75,G-100,& G-200)
2.Sepharose CL-series(2B,4B,6B).
3. Sephacryl(S-100,S-300).
4.Bio-gel
5.Silica gel
Applications:
The gel filtration chromatography is routinely used in several bio-medical and
research laboratories
1. To fractionate proteins,carbohydrates,nucleic acids and enzymes.
2. to fractionate cell organelles,viruses etc.,
3. To remove co-factors, inhibitors etc., from enzymes
4. To desalt the biomolecules before lyophilization or concentration or further
purification.
5. To remove free and low moleculres weight labels such as 125I,FITC from the
solution of labelled proteins.
7. Adsorption chromatography:
Ion-exchange chromatography
This techniques involves separation of biomolecules depending on their net
charges. In gel filtration techniques, a mixture of biomolecules are separated
based on molecular size, but the different biomolecules having identical or similar
molecules weight or size cannot be effectively separated. Such possibilities are
significant because many unrelated biomolecules may be identical in their
molecules weight or size. To solve this kind of problems ,other characteristics of
biomolecules such as net charge iso-electric point or their specific binding ability
are being utilized to separate the unrelated biomolecules having identical or
similar molecular weight.
APPLICATIONS:
The ion-exchange chromatography in mostly used in certain bio-medical and
research laboratories to
1. Separated many blood product such as albumin,IgG,plasma b-endorphins.
2. Separate products of recombinant DNA technology.
3.Separate T3 and T4.
4. Separate renal proteinuria in urine.
8. Affinity Chromatography
Affinity chromatography is a type of adsorption chromatography in which the
biomolecules or specific type of cells to be purified is specifically and reversibly
adsorbed by a complementary binding substances (Ligand) which is immobilized on
a insoluble support (matrix). It occupies a unique place in separation technology
since it is the only techniques which enables purification of almost any biomolecules
on the basis of its biological function or individual chemical structure .In addition
purification of a biomolecules is often in the order of several thousand fold and
recovery of active materials are generally very high and being a single step
purification it is an immense time saver over the other multi-stage purification
procedures.
Applications:
1.To purify enzyme using substrate as ligands.
2. To purify hormones using cell menbrance receptors.
3.To purify specific antibodies using antigens.
4.To purify mRNA using complementary DNA.
5.To purify interferons, viral RNA,etc.
