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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
As a general rule, a good H value is about two to three times the average particle diameter of the medium being packed. For a 90 µm particle, this means an H value of 0.018–0.027 cm.
The symmetry factor (As) is expressed as:As = b/awherea = 1st half peak width at 10% of peak heightb = 2nd half peak width at 10% of peak heightAs 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.
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 µmPreparative Separations 10-40 µmLow-pressure/large-scale applications 40-150 µmVery large scale operations ~300 µm
Ion Exchange ChromatographyIon Exchange Chromatography
Gel filtration Hydrophobic Ion Exchange Affinity Chromatography
•Biomolecules are purified using chromatography techniques that separate them according to differences in their specific properties.
•Ion exchange chromatography (IEX) separates biomolecules according to differences in their net surface charge.
Every technique offers a balance between resolution, capacity, speed and recovery.
•IEX for the separation of biomolecules was introduced in the 1960s and continues to play a major role in the separation and purification of biomolecules.
•Today, IEX is one of the most frequently used techniques for purification of proteins, peptides, nucleic acids and other charged biomolecules, offering high resolution and group separations with high loading capacity.
•The technique is capable of separating molecular species that have only minor differences in their charge properties, for example two proteins differing 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 from microscale purification and analysis through to purification of kilograms of product.
Ion-Exchange Chromatography
Ion Exchange Chromatography (IEX)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 purificationIEX Chromatography is the main means of protein purificationboth at laboratory and industrial scaleboth at laboratory and industrial scale
• IEX matrices are relatively cheapIEX matrices are relatively cheap
• IEX matrices have very high capacity (up to 100 mg protein perIEX matrices have very high capacity (up to 100 mg protein per ml gel) ml gel)
• IEX-chromatography has high resolutionIEX-chromatography has high resolution
• Versatile, the same column can be used for purification of Versatile, the same column can be used for purification of different proteinsdifferent proteins
• Optimization and scale up is rather straightforwardOptimization 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.
Ion Exchange Matrices- Functional groups Ion Exchange Matrices- Functional groups
Anion ExchangersAnion Exchangers Functional groupFunctional group--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Quaternary ammonium (Q) strong -O-CHQuaternary ammonium (Q) strong -O-CH22NN++(CH(CH33))33
Diethylaminoethyl (DEAE) weak -O-CHDiethylaminoethyl (DEAE) weak -O-CH22CHCH22NN++H(CHH(CH22CHCH33))22
Diethylaminopropyl (ANX) weak -O-CHDiethylaminopropyl (ANX) weak -O-CH22CHOHCHCHOHCH22NN++H(CHH(CH22CHCH33))22
Cation Exchangers Functional groupCation Exchangers Functional group--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Sulfopropyl (SP) strong -O-CHSulfopropyl (SP) strong -O-CH22CHOHCHCHOHCH22OCHOCH22CHCH22CHCH22SOSO33
- -
Methyl sulfonate (S) strong -O-CH-O-CH22CHOHCHCHOHCH22OCHOCH22CHOHCHCHOHCH22SOSO33--
Carboxymethyl (CM)Carboxymethyl (CM) weak -O – CH2COO-
___________________________________________________________________________
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).
Titration curves show the ion exchange capacity of strong ion exchangers Q and S.
Approximately 5 ml of Q or S Sepharose Fast Flow are equilibrated in 1 M KCl and titrated with 0. 1 M NaOH.
Strong Ion-Exchangers
Weak Ion-Exchangers
Anion or Cation Exchanger?
Protein A
Protein B
Protein C
Elution profile of three different proteins at various pHElution profile of three different proteins at various pH
The pH vs. net surface charge curves for three different proteins are shown. Schematic chromatograms for a CM and a DEAE ion exchanger are shown at the top and bottom respectively. At the most acidic pH value, all three proteins are positively charged and adsorb only to the CM ion exchanger. They are then eluted 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 will consequently adsorb to a DEAE ion exchanger, but not to a CM ion exchanger the other two do. At the next 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. At the most alkaline pH, all three proteins are adsorbed to the DEAE ion exchanger only. Thus, by varying
the pH of the mobile phase, one can greatly influence the selectivity in ion exchange chromatography.
Ion Exchange Matrices Ion Exchange Matrices
Trade name Material Mean particle size___________________________________________________________MiniBeads Polystyrene/divinyl benzene 3 µm
MonoBeads ----do--- 10 µm
SOURCE 30 ---do--- 30 µm
Sepharose High Performance Agarose 6% 34 µm
Sepharose Fast Flow Agarose 6% 90 µm
Sepharose 4 Fast Flow Agarose 4% 90 µm
Sepharose XL Agarose 6%, dextran chains 90 µm coupled to agarose
Sepharose Big Beads Agarose 6% 200 µm___________________________________________________________________
MonoBeads showing spherical, monodispersed particles.
Structure of cross-linked agarose media (Sepharose).
Uniform size distribution of SOURCE monodispersed particles.
Packed columns and resins
Elution profile with high salt concentrationElution profile with high salt concentration
Practical considerations for IEX separation
1. 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 effects
Mass transfer and diffusion effects
