MODERN SEPARATION

PROCESSES
Unit III – Separation by Adsorption
 CHROMATOGRAPHY
• Chromatography: physically separating
mixtures of gases, liquids and dissolved
substances
• Used to identify drugs, poisons, enzymes,
cells, proteins
• Separation is by melecular size and/ or
charge
• Separation is due to the differences in the
distribution coefficients of components
between two phases
 Concept
• Involves two phases
1. Stationary phase- Adsorbent phase:
material on which separation takes
place
2. Mobile phase- Solvent transports
the sample
 Types

• Gas Chromatography
• Liquid Column Chromatography
• High Performance Liquid Chromatography
(HPLC)
• Thin Layer Chromatography (TLC)
• Paper Chromatography
• Electrophoresis
• Affinity Chromatography
• Immuno chromatography
 Gas Chromatography
• Gas separates the components
• The components are then ionized
and an electric signal is recorded
• Mobile phase: Carrier gas
• Stationary phase: Thin film of liquid
 HPLC
• A liquid phase (mobile phase) is
pumped into a column containing the
stationary phase.
• Done at room temperature
 TLC
• A plate is coated with a granular gel
(silica gel or Aluminium oxide)-
Stationary phase
• The substance to be separated is
carried up by capillary action
• Substance with most affinity for the
plate will rise the farthest
 Paper Chromatography
• Same as TLC, but paper is used as
the stationary phase
 Electrophoresis
• Same as TLC but the substance is
separated through a gel by electric
current
• Due to different size and charge,
components will move across the
phase at different speeds
 Affinity Chromatography
• Binding specificity of biomolecules. Enzymes,
hormones, receptors, antibodies, antigens, binding
proteins, lectins, nucleic acids, vitamins, whole cells
and other components capable of specific and reversible
binding are amenable to highly selective affinity
purification.
• Column packing is prepared by linking a binding molecule
called ligand to an insoluble support
• When sample is passed through the column, only solutes
with appreciable affinity for the ligand are retained.
• Molecules called spacer arms are often used to set the
ligand away from the support and make it more accessible
to the solute.
• Many ready-made support-ligand preparations are
available commercially and are suitable for many ranges of
proteins.
 Immuno Chromatography
• Affinity chromatography using
antibody ligands is called Immuno-
affinity chromatography or Immuno-
chromatography.