Chromatography,

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Chromatography

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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Chromatography Components
Solutes which interact differently with the
stationary phase can be separated.
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Continue Developing with Solvent
Apply Sample
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Common Media Used in Liquid Protein Chromatography
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Chromatography
Generic Protocol 1. Prepare Column (?) 2. Apply
Sample 3. Wash 4. Elute 5. Analyze Fractions

• Equipment
• batch-wise
• home made
• work stations
• FPLC/HPLC

HPLC High Performance (Pressure) Liquid
Chromatography FPLC Fast Protein Liquid
Chromatography
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Ion Exchange Chromatography (IEC)

• based on charge-charge interactions between solid
  matrix and solute

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Basic Principal of IEC
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increasing formate ion concentration ?
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• Prepare or purchase column
• Adjust pH and initial counter ion
• Apply sample

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Elution from IEC Column

• change pH
• increase counter-ion (ie, salt) concentration
• in steps (eg, 0.1, 0.2, 0.3, 0.4 M NaCl)
• gradually (eg, 0?0.4 M NaCl) with gradient maker

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• collect fractions as column elutes
• analyze fractions for components of interest

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Hydrophobic Interaction Chromatography (HIC)

• separates proteins based on differences in
  hydrophobicity

• absorb proteins to hydrophobic matrix
• high salt promotes hydrophobic interactions
• eg, 1 M (NH4)2SO4

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Reverse Phase Chromatography

• separation based on total hydrophobicity
• generally used to separate small peptides

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Gel Filtration

• separation based on size, aka
• molecular sieve chromatography
• size exclusion chromatography
• media composed of cross-linked polymers
• pore size of matrix determines degree of
  interaction
• larger molecules are excluded and migrate faster
• smaller molecules are included and are retained
  longer

• Dextran (Sephadex)
• Agarose (Sepharose)
• Polyacrylamide

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• Applications
• purification
• desalting
• size determination

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Calculating Size
Vo void volume Vt total volume Ve elution
volume

• use size standards
• (relative MW)
• migration also affected by shape

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Affinity Chromatography

• based on specific binding of protein to ligand
• ligands can include
• substrate analogs, inhibitors
• natural ligands
• co-factors, metals
• binding proteins
• antibodies

• Elution destabilize binding
• compete with free ligand
• change pH, ionic strength
• chaotropic or denaturing agents