M.PRASAD NAIDUMsc Medical Biochemistry,
Ph.D Research scholar.
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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Continue Developing with Solvent
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?
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
Ion Exchange Chromatography (IEC)• based on charge-charge interactions
between solid matrix and solute
Basic Principal of IEC
increasing formate ion concentration
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
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
•collect fractions as column elutes
•analyze fractions for components of interest
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
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
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
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
Calculating Size
Vo = void volumeVt = total volumeVe = elution volume
Ve - Vo
Vt - VoKav =
• use size standards• (relative MW)• migration also
affected by shape
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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