Chromatography

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CHROMATOGRAPHY

• Presented by
• Mr.Halavath Ramesh
• M.A,M.sc,B.ED,PGDCAQM,PGDCA,M.Phil,(P.HD)(UoH)
• E-mail halavathramesh39_at_gmail.com
• University of Madras
• Dept. of Chemistry
• Loyola College ,Chennai.

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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

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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.

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• 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
• Solvent should be stable in air and when mixed
  with small quantities acid or alkaline vapor.
• Components of the mobile phase should be
  relatively non-volatile or their volatilability
  should be similar to the closed apparatus.
• The solvent should be removed rapidly and
  completely from the stationary phase after the
  chromatogram has been completed.
• The solvent should remain homogenous throughout
  the range of temperature experienced in
  laboratory.
• 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
  specific staining techniques such as ninhydrin
  for amino acid ,silver nitrate for reducing
  sugars and for lipids. After staining ,Rf values
  of each spot could be determined.

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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
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• 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
• To fractionate proteins,carbohydrates,nucleic
  acids and enzymes.
• to fractionate cell organelles,viruses etc.,
• To remove co-factors, inhibitors etc., from
  enzymes
• To desalt the biomolecules before lyophilization
  or concentration or further purification.
• To remove free and low moleculres weight labels
  such as 125I,FITC from the solution of labelled
  proteins.

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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. APPLICATIO
NS 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.
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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.
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