Rate Theory• Based on a random walk mechanism for the migration of molecules through a column• takes into account: – band broadening – effect of rate of elution on band shape – availability of different paths for different solute molecules to follow – diffusion of solute along length
The symmetry factor (As) is expressed as: As = b/a where a = 1st half peak width at 10% of peak height b = 2nd half peak width at 10% of peak height As should be as close as possible to 1. A reasonable As value for a short column as used in IEX is 0.80–1.80.As a general rule, a good H value is about two to three times theaverage particle diameter of the medium being packed. For a 90 µm particle,this means an H value of 0.018–0.027 cm.
MATRIX •Rigid Solids: Based on silica matrix, with stand high pressures (4000-6000 psi) •Hard gels: based on highly porous/non porous particles of polystyrene cross-linked with divinyl benzene • Soft gels: Such as cellulose/agarose, dextran, polyamide and other hydrophilic polymers. Separation of proteins etc. Typical Particle size used in Chromatography Analytical Applications 3-10 µm Preparative Separations 10-40 µm Low-pressure/large-scale applications 40-150 µm Very large scale operations ~300 µm
•Biomolecules are purified using chromatography techniques thatseparate them according to differences in their specific properties.•Ion exchange chromatography (IEX) separates biomolecules accordingto differences in their net surface charge. Gel filtration Hydrophobic Ion Exchange Affinity Chromatography
Every technique offers a balance between resolution, capacity,speed and recovery.
Ion-Exchange Chromatography•IEX for the separation of biomolecules was introduced in the1960s and continues to play a major role in the separation andpurification of biomolecules.•Today, IEX is one of the most frequently used techniques forpurification of proteins, peptides, nucleic acids and other chargedbiomolecules, offering high resolution and group separations withhigh loading capacity.•The technique is capable of separating molecular species that haveonly minor differences in their charge properties, for example two proteinsdiffering by one charged amino acid.•These features make IEX well suited for capture, intermediate purification orpolishing steps in a purification protocol and the technique is used frommicroscale purification and analysis through to purification of kilogramsof product.
Ion Exchange Chromatography (IEX)• IEX separates molecules on the basis of differences in their net surface charge.• Molecules vary considerably in their charge properties and will exhibit different degrees of interaction with charged chromatography media according to differences in their overall charge, charge density and surface charge distribution.• A protein that has no net charge at a pH equivalent to its isoelectric point (pI) will not interact with the charged medium.• At a pH above its isolectric point, a protein will have net negative surface charge and will bind to positively charged medium or anion exchanger• At a pH below its isolectric point, a protein will have net positive surface charge and will bind to negatively charged medium or cation exchanger
IEX Chromatography is the main means of protein purificationboth at laboratory and industrial scale• IEX matrices are relatively cheap• IEX matrices have very high capacity (up to 100 mg protein per ml gel)• IEX-chromatography has high resolution• Versatile, the same column can be used for purification of different proteins• Optimization and scale up is rather straightforward
Isoeletric point (pI) and Ion Exchangers• At a pH above its isoelectric point, a protein will bind to a positively charged medium or anion exchanger.• At a pH below its pI, a protein will bind to a negatively charged medium or cation exchanger.
STRONG/WEAK – What does it mean?• strong and weak do not refer to the strength with which the functional groups bind to proteins.• Strong ion exchangers show no variation in ion exchange capacity with change in pH.• These exchangers do not take up or lose protons with changing pH and so have no buffering capacity, remaining fully charged over a broad pH range.• Strong ion exchangers include Q (anionic), S and SP (cationic) (pH 2 - 12).• Weak ion Exchangers: DEAE (anion exchange) and CM (cation exchange) are fully charged over a narrower pH range (pH 2 - 9 and pH 6 - 10, respectively).
Strong Ion-ExchangersTitration curves show the ion exchange capacity of strong ion exchangersQ and S.Approximately 5 ml of Q or S Sepharose Fast Flow are equilibrated in1 M KCl and titrated with 0. 1 M NaOH.
Elution profile of three different proteins at various pH Protein A Protein B Protein CThe pH vs. net surface charge curves for three different proteins are shown. Schematic chromatogramsfor a CM and a DEAE ion exchanger are shown at the top and bottom respectively. At the most acidic pHvalue, all three proteins are positively charged and adsorb only to the CM ion exchanger. They are theneluted in the order of net charge. At the next pH value chosen, the protein has passed its isoelectric point and is now negatively charged, while the other two still retain positive charges. The blue protein willconsequently adsorb to a DEAE ion exchanger, but not to a CM ion exchanger the other two do. At thenext highest pH value the only one positively-charged protein still adsorbs to the CM ion exchanger.Because of their negative net charges, the two other proteins adsorb to the DEAE ion exchanger. Atthe most alkaline pH, all three proteins are adsorbed to the DEAE ion exchanger only. Thus, by varyingthe pH of the mobile phase, one can greatly influence the selectivity in ion exchange chromatography.
Ion Exchange MatricesTrade name Material Mean particle size___________________________________________________________MiniBeads Polystyrene/divinyl benzene 3 µmMonoBeads ----do--- 10 µmSOURCE 30 ---do--- 30 µmSepharose High Performance Agarose 6% 34 µmSepharose Fast Flow Agarose 6% 90 µmSepharose 4 Fast Flow Agarose 4% 90 µmSepharose XL Agarose 6%, dextran chains 90 µm coupled to agaroseSepharose Big Beads Agarose 6% 200 µm___________________________________________________________________
MonoBeads showing spherical, Uniform size distribution of SOURCE monodispersed particles. monodispersed particles.Structure of cross-linked agarosemedia (Sepharose). Packed columns and resins
Practical considerations for IEX separation1. Equilibrate column with 5–10 column volumes of start buffer or until the baseline, eluent pH and conductivity are stable.2. Adjust the sample to the chosen starting pH and ionic strength and apply to the column.3. Wash with 5–10 column volumes of start buffer or until the baseline, eluent pH and conductivity are stable i.e. when all unbound material has washed through the column.4. Begin elution using a gradient volume of 10–20 column volumes with an increasing ionic strength up to 0.5 M NaCl (50%B).5. Wash with 5 column volumes of 1 M NaCl (100%B) to elute any remaining ionically bound material.6. Re-equilibrate with 5–10 column volumes of start buffer or until eluent pH and conductivity reach the required values.
Band broadening effectsMass transfer and diffusion effects