Chromatography

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CHROMATOGRAPHY

• M.PRASAD NAIDU
• Msc Medical Biochemistry,
• Ph.D Research scholar.

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• CHROMATOGRAPHY
• Historical details
• 1903 Michael Tswett (or Tsvet) a Russian
  botanist separated the pigments in green plant
  leaves using a glass column packed with 2 µm
  inulin and ligroin as mobile phase
• Coined the term chromatography - color writing -
  to describe the separation of the pigments into
  colored bands along the length of the column
• Later used CaCO3 or sucrose as the column packing
  material
• Not much work done until the late 1930s and
  1940s
• 1941 Archer John Porter Martin Richard
  Laurence Millington Synge published a paper on
  liquid partition chromatography that set the
  stage for gas liquid chromatography
• Studied the amino acid composition of wool
• Invented a process where a liquid is firmly bound
  to a finely granulated solid phase packed in a
  glass column
• H2O firmly bound to SiO2 - silica gel
• A second liquid immiscible with the bound H2O is
  percolated through the column - CHCL3
• Solute analyte molecules are partitioned between
  the stationary phase and the mobile phase

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• Historical details
• Partition chromatography
• Problem the packing of silica gel columns is not
  very reproducible in its properties
• Martin and Synge invented a new method in which
  H2O is firmly bonded to filter paper
• Solute analyte molecules are partitioned between
  a moving liquid phase and the H2O bound to the
  cellulose surface
• Very reproducible elution times are obtained
  using paper chromatography
• 1952 Martin and Synge were awarded the Nobel
  prize in chemistry for the invention of column
  partition chromatography, and particularly paper
  chromatography
• 1938 Izmailov Schreiber worked out a procedure
  where the solid stationary chromatographic
  phase is distributed as a thin film on a glass
  plate
• Thin Layer Chromatography (TLC) was not much used
  until 1956 when it began to be used

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• Chromatography
• Historical details
• Gas chromatography was invented in Austria but
  was not much used until 1956
• 1956 Martin James applied gas chromatography
  to the separation of acids and amines
• Small particles are coated with a non-volatile
  liquid and packed into a tube which can be
  heated
• Analytical mixtures are injected into a heated
  inlet tube and volatilized
• The mixture is driven through the packed column
  by a compressed gas
• Interactions between the components of the
  mixture and the liquid stationary phase causes
  separation into zones as the analytes are passed
  through the column
• Major recent advances involve the elimination of
  the packing
• Coat the inside of a fused silica capillary - 250
  µm ID - tube with a liquid film - 0.10 - 0.50
  µm thickness
• Most recently, 530 µm ID tubes - megabore
  capillaries - are being used

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• Chromatography
• Definition of chromatography The differential
  migration of sample components dissolved in a
  mobile phase produced by selective retention by a
  stationary phase
• Classification of chromatographic methods
• Chromatography
• Mobile Phase Gas Liquid
• Stationary
• phase Liquid Solid Bonded Liq.
  Liq Solid Bonded Liq. Ion Ex Polymer
  
• 
  
  Solid
• Process (partn) (adsorpn) (adsorpn-
  (partn) (adsorpn) (adsorpn- (ion
  (partn
• 
  partn) HPLC
  partn) excng) sieving)
• 
  paper
  HPLC exclusion
• 
  
  chromat
• Conditions associated with each kind of
  chromatography
• Gas-liquid partition chromatography involves
  establishing an equilibrium between components
  in the gaseous mobile phase and the liquid
  stationary phase

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• Chromatography
• Conditions associated with each kind of
  chromatography
• Gas-solid adsorption chromatography involves
  surface adsorption of analytes dissolved in the
  gas mobile phase using solids such as alumina,
  molecular sieves or porous silica
• Gas bonded-liquid chromatography makes use of
  molecules that are chemically bonded to a solid
  support, either silica beads or the wall of a
  fused silica capillary tube
• The interaction between components dissolved in
  the stationary phase and the stationary phase
  involves both partitioning and adsorption
• Liquid-liquid partition chromatography involves a
  partitioning of components dissolved in the
  mobile phase between the mobile phase and the
  stationary liquid phase
• This is the process often associated with High
  Performance Liquid Chromatography (HPLC), paper
  chromatography and TLC if silica gel is used
• Liquid-solid adsorption chromatography involves
  an adsorption of analytes dissolved in the
  liquid mobile phase on the surface of the
  stationary phase the stationary phase can be
  any adsorbent material such as alumina or silica

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• Chromatography
• Conditions associated with each kind of
  chromatography
• Liquid bonded-liquid chromatography involves
  silica beads (3-5 µm diameter) to which
  molecules are chemically bonded
• The interaction between analyte molecules in the
  liquid mobile phase and the stationary phase
  involves both partitioning and adsorption
• This is now the usual method for carrying out
  HPLC
• Normal phase HPLC uses a polar stationary phase
  and a non-polar liquid mobile phase
• Reversed phase HPLC uses a non-polar stationary
  phase and a polar liquid mobile phase
• Ion exchange chromatography makes use of natural
  or synthetic zeolites or synthetic or organic
  or inorganic polymer resins as the stationary
  phase
• The interaction between ions dissolved in the
  liquid mobile phase and the stationary phase
  involves an exchange of ions associated with the
  stationary phase

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• Chromatography
• Conditions associated with each kind of
  chromatography
• Ion exchange chromatography
• Organic cation exchange resins involve
  crosslinked polystyrene containing either SO3-
  or COO- functional groups with an associated
  cation
• Organic anion exchange resin involve
• crosslinked polystyrene containing NH3
• functional groups with an associated anion

The affinity of dissolved ions for the resin
varies with the ion and the composition of the
solution
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• Chromatography
• Conditions associated with each kind of
  chromatography
• Exclusion chromatography makes use of a uniform,
  highly porous, non-ionic gel
• Small molecules are retained in the pores of the
  gel, large molecules are not retained - they are
  excluded
• Separation involves sorting molecules by size
• Gel permeation chromatography involves use of
  polymers that swell in organic liquids -
  polyacrylamide gels
• Gel filtration chromatography involves the use of
  polymers that swell in water such as Sephadex, a
  polysucrose polymer
• For liquid chromatography, differences in the
  affinity of solute analytes for the stationary
  phase can be controlled by controlling the
  physical and chemical properties of not only the
  stationary phase but also the mobile phase
• Gradient elution liquid chromatography involves
  changing the composition of the mobile phase
  during the elution process
• Such liquid properties as the polarity, ionic
  strength and pH can be varied
• Isocratic liquid chromatography involves constant
  mobile phase composition

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• Chromatography
• Chromatographic column theory of packed columns
• The differential migration of analytes in the
  mobile phase separates components into zones
  that move at different rates along the column
  length

• Observations
• Bands move down the column with different
  velocities relative to the mobile phase front
• Zones get wider the longer they remain in the
  column
• There is a normal distribution of molecule
  concentration along the length of the column

t0 t1 t3 t4 t5
2 components
AB mobile phase front
detector
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• Chromatography
• Chromatographic column theory of packed columns
• Resolution measures how well zones are separated
  compared to their overlap
• Determined by the standard deviation (s) in
  column length over which the bands are
  distributed
• Determined by the retention time how long it
  takes a zone center to elute

• W is determined by column efficiency
• Function of flow rate, packing particle size,
  column diameter
• ?tR determined by column selectivity
• Function of the nature of the mobile and
  stationary phases
• Function of relative amounts of mobile and
  stationary phases
• Function of how efficiently the stationary
  phase is used as measured by the capacity
  factor - k

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• Chromatography
• Chromatographic column theory of packed columns
• The effect of column efficiency and column
  selectivity on resolution

Poor resolution because of poor column
efficiency Good resolution because of good
column efficiency, although column selectivity
is not great Good resolution because of good
column selectivity, although column efficiency
is poor Poor resolution because of poor column
selectivity, although column efficiency is good
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