1. 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

4. 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 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.

5. 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

6. Ion Exchange Chromatography

7. •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.
Gel filtration Hydrophobic Ion Exchange Affinity Chromatography

8. Every technique offers a balance between resolution, capacity,
speed and recovery.

9. Ion-Exchange Chromatography
•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 or
polishing steps in a purification protocol and the technique is used from
microscale purification and analysis through to purification of kilograms
of product.

10. 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

11. IEX Chromatography is the main means of protein purification
both 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

12. 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.

13. Ion Exchange Matrices- Functional groups
Anion Exchangers Functional group
----------------------------------------------------------------------------------------------------
Quaternary ammonium (Q) strong -O-CH2N+(CH3)3
Diethylaminoethyl (DEAE) weak -O-CH2CH2N+H(CH2CH3)2
Diethylaminopropyl (ANX) weak -O-CH2CHOHCH2N+H(CH2CH3)2
Cation Exchangers Functional group
----------------------------------------------------------------------------------------------------------------
Sulfopropyl (SP) strong -O-CH2CHOHCH2OCH2CH2CH2SO3-
Methyl sulfonate (S) strong -O-CH2CHOHCH2OCH2CHOHCH2SO3-
Carboxymethyl (CM) weak -O – CH2COO-
___________________________________________________________________________

14. 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).

15. Strong Ion-Exchangers
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.

16. Weak Ion-Exchangers

17. Anion or Cation Exchanger?

18. Charge on Protein Depend on the IpH and pH of the Buffer.

19. Selectivity as it is Influence by pH.

20. Elution profile of three different proteins at various pH
Protein A
Protein B
Protein C
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.

21. 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
___________________________________________________________________

22. MonoBeads showing spherical, Uniform size distribution of SOURCE
monodispersed particles. monodispersed particles.
Structure of cross-linked agarose
media (Sepharose).
Packed columns and resins

23. Elution profile with high salt concentration

24. 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.

26. Band broadening effects
Mass transfer and diffusion effects