Separating proteins Ion chromatography

preparative-scale ion exchange column used protein purification.

ion exchange chromatography can used separate proteins because contain charged functional groups. ions of interest (in case charged proteins) exchanged ions (usually h) on charged solid support. solutes commonly in liquid phase, tends water. take example proteins in water, liquid phase passed through column. column commonly known solid phase since filled porous synthetic particles of particular charge. these porous particles referred beads, may aminated (containing amino groups) or have metal ions in order have charge. column can prepared using porous polymers, macromolecules on 100,000 optimum size of porous particle 1 μm. because slow diffusion of solutes within pores not restrict separation quality. beads containing positively charged groups, attract negatively charged proteins, commonly referred anion exchange resins. amino acids have negatively charged side chains @ ph 7 (ph of water) glutamate , aspartate. beads negatively charged called cation exchange resins, positively charged proteins attracted. amino acids have positively charged side chains @ ph 7 lysine, histidine , asparagine.

the isoelectric point ph @ compound - in case protein - has no net charge. protein’s isoelectric point or pi can determined using pka of side chains, if amino (positive chain) able cancel out carboxyl (negative) chain, protein @ pi. using buffers instead of water proteins not have charge @ ph 7, idea enables manipulation of ph alter ionic interactions between proteins , beads. weakly acid or basic side chains (such in leucine, proline, alanine, valine, glycine…etc.) able have charge if ph high enough deprotonate amino group. separation can achieved based on natural isoelectric point of protein. alternatively peptide tag can genetically added protein give protein isoelectric point away natural proteins (e.g., 6 arginines binding cation-exchange resin or 6 glutamates binding anion-exchange resin such deae-sepharose).

elution increasing ionic strength of mobile phase more subtle. works because ions mobile phase interact immobilized ions on stationary phase, shielding stationary phase protein, , letting protein elute.

elution ion-exchange columns can sensitive changes of single charge- chromatofocusing. ion-exchange chromatography useful in isolation of specific multimeric protein assemblies, allowing purification of specific complexes according both number , position of charged peptide tags.

gibbs-donnan effect

in ion exchange chromatography, gibbs–donnan effect observed when ph of applied buffer , ion exchanger differ, 1 ph unit. example, in anion-exchange columns, ion exchangers repeal protons ph of buffer near column differs higher rest of solvent. result, experimenter has becareful protein(s) of interest stable , charged in actual ph.

this effect comes result of 2 charged particles, 1 resin , 1 solution, failing distribute between 2 sides; there selective uptake of 1 ion on another. example, in sulphonated polystyrene resin, cation exchange resin, chlorine ion of hydrochloric acid buffer should equilibrate resin. however, since concentration of sulphonic acid in resin high, hydrogen of hcl has no tendency enter column. this, combined need of electroneutrality, leads minimum amount of hydrogen , chlorine entering resin.