Gel chromatography Gel permeation Molecular sieving Size exclusion steric exclusion

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Gel chromatographyGel permeationMolecular
sievingSize exclusion steric exclusion
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Principle of separation

• It is a kind of chromatography technique based on
  the difference of molecular weight and is one of
  the effective and mild methods extensively used
  to isolate and analyze the biomacromocular
  substances.
• The stationary phase consists of beads containing
  pores that span a relatively narrow size range.
• when the gel is packed into a column and
  percolated with a solvent, it permits the large
  molecular weight compounds to pass rapidly
  without penetration of the pores
• Smaller molecules spend more time inside the
  beads than larger molecules and therefore elute
  later (after a larger volume of mobile phase has
  passed through the column).

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Nature of the gel

• 1- chemically inert
• 2- mechanically stable
• 3- ideal porous structure
• Wide pore size give low resolution
• 4- uniform particle size
• Types of gel

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Types of gel

• 1- Sephadex
• ? 1-6-polymer of glucose is prepared by microbial
  fermentation of sucrose (glucose fructose)
• The resulting glucose provids the required a1-6
  glucosan polymer called dextran
• The resulting dextran is treated with
  epichlorohydrin to give several types of crossed
  linked dextran (sephadex)

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Cross linking
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• Sephadex is obtained in different degrees
  depending on the pore size
• High percentage of epichlorohydrin give high
  degree of cross linking (small pore size)
• Lower percentage produce sephadex with large pore
  size
• Characters of sephadex
• 1- highly stable gels
• 2- stable at PH 2-12
• 3- their particles are free from ions
• 4- insoluble in water and organic solvent
• 5- they swell in water and other hydrophillic
  solvent
• 6- they require bactericidal such as Hg acetate

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• 2- Agarose gel
• Obtained from agar and composed of alternating
  units of 1,3 linked ß-D-gal and 1,4 linked
  3,6-anhydro-a, L-galactose
• This was subjected to epichlorohydrin to give
  sepharose
• Characters
• 1- it dissolves in H2O at 50 c and on cooling
  form gel
• 2- insoluble below 40 c
• 3- freezing destroys the gel

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• 3- Acrylamide gels 9synthetic gel)
• It is not dextran polymer
• It is polymerized acrylamide or
  methylen-bis-acrylamide

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• Column packing
• 1- gel is mixed with solvent for 3 hrs to swell
• 2- pack the column
• 3- sample should be solution
• 4- Not to allow dry

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• Application of gel filtration chromatography
• 1- separation of large molecular weight compound
  as protein, carbohydrate, peptides, nucleic acids
• 2- desalting of colloids
• Small size of contaminating salt will allow them
  to diffuse inside the gel particles
• E.g. Desalting of albumin from 25 ammonium
  sulfate
• 3- molecular weight determination
• A linear relationship exists between the
  logarithm of the molecular weight and the elution
  volume

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Ion exchange chromatography

• Mix-X- RY- Y- RX- anionic
  exchange
• Mix-X R-Y Y- R-X cationic
  exchange
• The polymer matrix carries functional groups that
  carries a positive or negative charge (fixed
  charge), which is balanced by ions of opposite
  charge (counter ions) these counter ion is
  lossely attached to the matrix and can change
  places with ions similar charge in solution

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• Advantages
• 1- separation of very pure compound from extract
• 2- require small amount of solvent
• 3- very useful in microbial fermentation for
  antibody production

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• Anaion exchange as
• strong anion as quaternary ammonium form Matrix-
  (NR3) -Cl-
• - weak anion as Matrix-NH2(CH3)-Cl-
• Cation exchange as
• sulfonic acid Matrix-(SO3) H (strong).
  
• And Matrix-COO- H (weak)
• The stronger the charge on the sample, the
  stronger it will be attached to the ionic surface
  and thus the longer it will take to elute.
• The mobile phase is an aqueous buffer, where the
  PH is adjusted to control elution time

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• Sulphonic acid (Cation)
• Quaternary ammonium group (Anion)

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• Substance form ion in aqueous solution (carry
  charge) when they are brought into contact with
  the head of ion exchange interaction occurs .
• The ion exchange expel or repels ions carrying
  the same charge as the fixed charge and will bind
  ion of the opposite charge.
• The beads of the ion exchangers represent the
  stationary phase and the solution following
  through is the mobile phase.

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Types and preparation of exchange material

• A) Ion Exchange Resins
• 1- Cross linked cation exchange resins
• Condensation of polyhydric phenols with
  formaldehyde to give uni-functional resins
  charged by the exchangeable H of the phenolic OH
• Now it is prepared by copolymerization between
  styrene and divinyl benzene and then sulfonic
  acid groups were introduced by sulfonation

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• cation exchange
• Strong cation
• Weak cation exchange can be prepared by
  copolymerization of methacrylic acid with divinyl
  benzene

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• Weak cation resin
• 2- anion exchange resins
• They are prepared in similar way to that of
  anion using cross linked polystyrene which is
  chloromethylated which is then treated with a
  secondary amine to give weakly basic tertiary
  amine resin or with primary amine to give weak
  anion exchanger

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• Strong basic quaternary amine resin
• Weak anion

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• B) ION EXCHANGE GELS
• Dextran (sephadex, cross linked dextran)
  inorganic unit introduced on the cross-linked
  dextran (sepahdex) by estrification of the
  hydroxyl groups by reagent contains terminal acid
  or base
• E.g. Sulphoxyl SO3H strong H
• Carbomethoxy CH2-CCO weak H
• Diethyl amino ethyl (DEAE) weak base
• C) ION EXCHANGE CELLULOSE

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Factors affecting the exchange potential

• 1- the valence of the exchanging ion Ca more than
  Na
• 2- increase with atomic number
• Li less than Na , Ca
• 3-the exchange of H or OH depends on the
  strength of the acid or the base formed with the
  functional group of the resin
• The weaker the acid or base formed the greater
  the exchange potential

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Ion exchange techniques

• 1- batch technique
• 2- column technique
• 1- Batch technique
• The resin is allowed to contact with the solution
  in a vessel and equilibrium is reached by shaking
  or stirring

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• 2- column technique
• The most common types
• 1- washing the resin is washed with mobile phase
  for the purification of degradation product from
  industry
• 2- swelling leave resin for 10-20 min in mobile
  phase to facilitate the softening of resin and
  facilitate penetration
• 3- sample application
• 5 g extract (in 20 ml solvent) added onto the top
  of a column then 0.5-1ml/min flow rate and
  collect fractions

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The effect of the PH on the capacity of ion
exchangers

• The capacity of the ion-exchanger resins is
  determined by the concentration of measurable
  ionic groups within the structure, The capacity
  of ion-exchangers is a function of PH
• Rcat.H R(-ve)cat. Hve     Equation
  1Ran.OH R(-ve)an. OH-ve   
  Equation 2
• Where Rcat. Ran. are cation anion exchangers,
  respectivelyIn equation 1 it is clear that the
  ionization of a cation exchange resin (Rcat.H) to
  produce the resin ion (R-ve cat.) H is
  influenced by PH. Thus at low PH (high
  concentration of hydrogen ions), the ionization
  of the acidic resin is inhibited the exchange
  capacity is decreasedIn equation 2, the
  ionization of the basic ion exchanger is
  inhibited at high PH, thereby reducing the
  exchange capacity of this resin
• So the PH will directly affect the ionization
  state of the resins either leading to increasing
  the resolution or decreasing it depending also on
  the functional groups the chemical nature of
  the resin itself

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Applications of IEC

• 1- analytical applications
• -water softening , exchange of Ca, Mg, Pb and Hg
  by Na
• 2- determination of total salt concentration
• RH salt (NaCl, unknown).RNa HCl (titrated
  with N/2 NaOH)
• 3- separation of interfering ions or electrolyte
• 4- Ion exclusion (Donnan exclusion) separation of
  electrolytes from non electrolytes

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Applications of IEC in the field of natural
products and pharmacy

• 1- separation of antibiotics
• 2- separation of vitamins
• 3- separation of amino acids
• 4- separation and purification of alkaloids