Broad Categories of Chromatography a) Ion exchange b) Gel

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Broad Categories of Chromatography

• a) Ion exchange
• b) Gel filtration/molecular sieving
• c) Affinity chromatography
• d) Reverse phase methods
• e) Hydrophobic interaction chromatography
• f) Chiral separations.

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Ion exchange

• Proteins have Charged groups on them
• His Lys Arg
• Glu Asp
• To a lesser extent Cys and Tyr
• The sum of the charges result in the protein
  behaving as an ion.
• Small molecules can have charge as well and be
  separated on that basis

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Ion exchange
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• Anion exchangers Functional group
• Diethylaminoethyl (DEAE)
• -O-CH2-CH2-NH(CH2CH3)2
• Quaternary aminoethyl (QAE)
• -O-CH2-CH2-N(C2H5)2-CH2-CHOH-CH3
• Quaternary ammonium (Q)
• -O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N(CH3)3
• Cation exchangers Functional group
• Carboxymethyl (CM)
• -O-CH2-COO
• Sulphopropyl (SP)
• -O-CH2-CHOH-CH2-O-CH2-CH2-CH2SO3-
• Methyl sulphonate (S)
• -O-CH2-CHOH-CH2-O-CH2-CHOH-CH2SO3-

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Ion exchange

• Elution
• Salt Gradients Salts increase the dielectric
  and screen charges from on another
• Step gradiants
• Linear gradients

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Ion exchange

• Elution pH Gradients Two mechanisms
• Ligand for Weak anion and cation exchangers the
  ligand will deionize as the pH changes as it
  deionizers the molecule elutes Strong cation and
  anion exchangers are charged at all reasonable pH
  regimes

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Ion exchange

• Elution pH Gradients Two mechanisms
• Molecule As the pH changes the charge on the
  molecule of interest changes below pI the
  molecule has a positive nature as the pH
  increases the molecule moves to neutral as it
  approaches the PI and then the molecule takes on
  a negative charge.

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Affinity chromatography

• Basic Idea
• Attach a Substrate/ligand on the media
• Run your molecule of interest over the media
  under binding conditions.
• Wash non-specific interactions off
• Elute by flooding with ligand/substrate or other
  condition that causes release.

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Affinity chromatography

• Multitude of media
• Heparin
• Poly Anionic (Sulfonated) Glucose.
• Mimics DNA to a certain extent
• Can be uses as a strong Cation exchange media
• Protein A, G
• Bind strongly to antibodies

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Affinity chromatograph

• Multitude of media
• Fusion techinques
• Chelated metal Chromotography
• Nickel Poly His tail fusion proteins
• Othe metals Trp His Cys on surface
• Biotin
• Avaden Fusion proteins
• Glutathione
• glutathione-S-transferase or GST fusion proteins

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Affinity chromatography

• substrate analogue, inhibitor, cofactor
• Enzymes
• Antibody
• antigen, virus, cell.
• Lectins
• polysaccharide, glycoprotein, cell surface
  receptor, cell.

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Affinity chromatography

• Nucleic acid
• complementary base sequence, histones, nucleic
  acid polymerase, nucleic acid binding protein.
• Hormone, vitamin
• receptor, carrier protein.

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Gel filtration/molecular sieving

• Usually a polishing step
• Also used to exchange buffers.
• Very non-specific.
• Separation based on size.
• Largest molecules move fastest.

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Hydrophobic interaction chromatography

• based on the interaction between hydrophobic
  patches on the surface of the moity of interest
  (typically protien) and the hydrophobic ligand on
  the support resin.

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Hydrophobic interaction chromatography

• Media (ligands)
• Phenyl Sepharose
• Butyl Sepharose
• Octyl Sepharose
• Chain length effects
• Degree of substitution

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Hydrophobic interaction chromatography

• Adsorption and elution are accomplished by
  changing the dielectric of the mobile phase.
• The more polar the tighter the binding
• The less polar the weaker the binding

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The Hydrophobic Interaction

• Really the Entropic Force
• DH for solvation of most hydrocarbons is
  Negative
• -TDS is positive and large for lower temps
• At higher temps water is disordered and -TDS
  starts to increase

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Hydrophobic interaction chromatography

• Changes in Dielectric
• Changes in Molecular Order
• Changes in Surface Tension
• Hofmeister Series
• NH4 gtKgtNagtLigtMggtCagtGuanadinium
• SO42-gtgtHPO42-gtAcetategtCitrategt TartrategtCl- gt
  NO3-gt ClO3-gtI-gtClO4-gtSCN-
• Disruptive gtorganizing
• increase surface tensiongt decrease

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d) Reverse phase methods

• Similar to HIC
• Higher ligand density
• 20 40 mm VS gt 200 mM/ml
• This requires lower dielectric to elute (More
  organic)
• Best for small molecules, peptides and small
  proteins that are resistant to organic
  de-naturation.

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d) Reverse phase methods

• Media
• C4
• C8
• C18
• Phenyl
• Usually silica based materials.

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d) Reverse phase methods

• Solvents
• Usually water/Methanol or water/acetonitrile
  mixture to elute.

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Chiral separations.

• Either the Media or the Mobile phase is Chiral
• Media can be
• Chiral ligands Cyclodextrans, small molecules,
  proteins
• Chiral structures helical polymers as media base
• Chiral mobil phases known.
• Mostly small molecule.

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