Chromatography

M.Prasad NaiduMSc Medical Biochemistry, Ph.D,.

Chromatography Components

stationary phase (eg., solid matrix)

mobile phase (eg., solvent) solute

Solutes which interact differently with the stationary phase can be separated.

ApplySample

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 Protocol1. 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 RPCMobile Phase Polar

SolventNonpolarSolvent

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