chromatography
DESCRIPTION
goodTRANSCRIPT
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M.PRASAD NAIDUMsc Medical Biochemistry,
Ph.D Research scholar.
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Chromatography Components
stationary phase (eg., solid matrix)
mobile phase (eg., solvent) solute
Solutes which interact differently with the stationary phase can be separated.
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ApplySample
Continue Developing with Solvent
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Common Media Used in Liquid Protein Chromatography
Media Type DiscriminationIon Exchange ChargeGel Filtration Size and ShapeHydrophobic Surface HydrophobicityReverse Phase Total HydrophobicityAffinity Specific Amino AcidsAdsorption Amino Groups?
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Chromatography
Generic Protocol
1. Prepare Column ()2. Apply Sample3. Wash4. Elute5. Analyze Fractions
Equipment• batch-wise• home made• work stations• FPLC/HPLC
HPLC = High Performance (Pressure) Liquid ChromatographyFPLC = 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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Amino a. pKa Asp, Glu 4.3-4.7 His 7 Lys, Arg > 10
• 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, 00.4 M NaCl) with
gradient maker
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•collect fractions as column elutes
•analyze fractions for components of interest
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increasing salting out effectanions: PO4, SO4, Cl, Br, NO3, Cl04, I, SCNcations: NH4, Rb, K, Na, Li, Mg, Ca, Ba
increasing chaotropic effect
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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HIC vs RPC
Mobile Phase PolarSolvent
NonpolarSolvent
Conditions Native DenaturedSoluteDiscrimination
SurfaceResidues
TotalResidues
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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SephadexCode Range (kDa)G-25 1-5G-50 2-30G-100 4-150G-150 5-300G-200 5-600
• choose matrix with desired characteristics• size range• does not interact with solute• include 0.15-1 M NaCl in buffer
• load sample in smallest possible volume• elute in one column volume
Practical Considerations
Applications:
• purification• desalting• size determination
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Calculating Size
Vo = void volumeVt = total volumeVe = elution volume
Ve - Vo
Vt - VoKav =
• 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
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THANK YOU