Principles of Ion -exchange chromatography, High performance liquid chromatography (HPLC) , chromatography generally stands for a technique which separates mixtures based on different dynamic sharing of their components between two distinct physio-chemical environments called mobile and stationary phase by repeated absorption/desorption steps. Ion chromatography (IC) is a member of large family of liquid phase chromatographic methods (that is a mobile phase is a liquid and a stationary phase is a solid).

1. Ion -exchange chromatography
They could not drink of the water of Marah, for they were
bitter...And he cried unto the LORD; and the LORD shewed
him a tree, which when he had cast into the waters, the
waters were made sweet...
Exodus, Chapter 15, versus 23-25

2. Introduction to chromatography
• separates mixtures by repeated absorption
/desorption steps
• components weakly held by the stationary
phase move quicker down the column
• difference in migration rates through the
column results cause distinct bands for sample
components

3. Classification of Chromatography based on the
mobile phase of the system (liquid or gas) and
stationary phase (liquid or solid)
• Liquid-liquid eg. HPLC
• liquid-solid, eg. silica column chromatography,
Ion chromatography (IC)
• gas-liquid, eg. GC
• gas-solid eg. Molecular sieves to purify gases

4. Classification of Chromatography based
on the physical equipment used.
• Column chromatography - flow through a
packed column.
• Paper chromatography - selective migration of
compounds across a plane of paper.
• Thin-layer chromatography - flow of a mobile
phase through a small gap created by two
planes of stationary material.

5. subclasses of column chromatography
based on column packing.
• Ion-exchange chromatography - ion-exchange
resin as the adsorbent.
• Gel chromatography - controlled porosity gel
as a packing.
• Affinity chromatography - certain proteins
separated by protein-ligand interactions.

6. Classification of ionic samples based
on ion chromatography
• Neutral sample- compounds that are neutral ,
eluted from the column without separation.
• Ionic samples - (acids, bases, organic salts)
• ionic solute with organic molecule in
protonization (base) or deprotonization (acid)
in the pH range 1 – 14.

7. Principles of Ion -exchange
chromatography
• Anions - separated on columns packed with an anion
exchange resin.
• Cations - separated on columns with cation exchange resin
• Anion exchangers carry a positive charge (quaternary
ammonium or amine groups) and separates anionic i.e. acidic
compounds.
• Cation exchangers –ve charge (sulfonate or carboxylate
groups) and separates cations i.e . protonated bases.

8. Functional groups attached on ion
exchangers
Anion exchangers Functional groups
diethyl amino ethyl (DEAE)
quarternary amino ethyl (QEA)
quarternary ammonium (Q)

9. Functional groups attached on ion
exchangers
Cation exchangers Functional groups
carboxymethyl (CM)
Sulphopropyl (SP)
Methyl sulphonate (S)

10. Principles of Ion -exchange
chromatography
• separation - reversible adsorption of charged
sample molecules to an ion exchanger (matrix) of
opposite charge.
• Adsorption organized by pH or ionic strength of
the eluting buffer.
• four staged mechanism
1. Equilibration
2. Sample Loading and Adsorption
3. Desorption or Elution
4. Regeneration

11. Equilibration- counter ions are
ionically bonded to matrix
• A stationary phase of an exchange resin covered
with counter ions from buffer with low ionic
strength.
• equilibrating the ion exchanger with the starting
buffer with desired pH and ionic strength.
• The pH for charged analyte, to bind resin

12. Sample adsorption – sample molecules
displace counter ions
• the sample loaded into the column
• charged sample molecules displace the counter ions
on the ion exchanger
• Chargeless unbound substances or the matrix
charged (ion exchanger washed through the column.

13. Elution – sample molecules are
displaced by ions from eluting buffer
• Attached solute molecules removed by
changing the elution conditions
• ionic strength or pH of eluting buffer.

14. Elution – sample molecules are
displaced by ions from eluting buffer
• Competition among salt ions (Na+ or Cl-) from
the buffer compete with the bound compounds
for the charged stationary phase.
• Retention of acidic or basic samples depends on
the pH value.
• The higher the ionization, the higher retention
will be.
• Lowering the pH will cause the analyte to be less
negatively-charged and less likely to interact with
the positively-charged anionic exchange resin.

15. Regeneration- matrix is regenerated
with original counter ions
• All the bounded impurities are eluted
(washed) from column
• the ion exchanger is regenerated with the
original counter ions.

16. Applications of Ion-exchange
chromatography
1. to isolate and purify protein samples
• For proteins, the charge is based on the protein's isoelectric point,
where the protein is neutrally charged (amphoteric), and measured
as pI
• At a pH above its pI, the compound will be negatively charged,
hence an anion exchange support is used, if stable at a pH above its
pI,
• at a pH below its pI, the compound will be positively charged,
hence an cation exchange support is used,
• Anionic exchange Chromatography is carried out with cationic
buffers.
• Cationic exchange Chromatography is carried out with anionic
buffers.
• The pK of the buffer is near to the pH at which the system is
buffered.

17. Applications of Ion-exchange
chromatography
1. to isolate and purify protein samples
• After sample adsorption, these can be eluted by
• Gradient Elution-if the sample components differ widely in
retention or if the adsorption of sample mixture is strong,
• elution is done by selectively decreasing the affinity of the sample
molecules for the charged groups on the ion exchanger.
• changing the composition, pH or ionic strength of the mobile phase
over a period of time.
• elution by changing the pH towards isoelectric point (where no
binding occurs) can desorb and elute the sample components from
the column
• by increasing the ionic strength gradually reduces the availability of
charged groups and elute the sample components from the column

18. Applications of Ion-exchange
chromatography
1. to isolate and purify protein samples
• Common buffers used

19. Applications of Ion-exchange
chromatography
2. water analysis
• Anion-exchange chromatography can be used to
measure the concentration of anions, including
sulfates, nitrates, nitrites, fluoride, and chloride.
• Cation-exchange chromatography is used to
measure the concentration of cations such as
sodium, potassium, calcium, and magnesium.
• magnesium and calcium ions in hard water can
be removed from water using ion-exchange
chromatography in water softeners by binding
them to a resin, which releases bound sodium.

20. References
• Jackson, Peter; Haddad, Paul R. (1990). Ion chromatography: principles and
applications. Amsterdam: Elsevier.
• Tatjana Weiss; Weiss, Joachim (2005). Handbook of Ion Chromatography.
Weinheim: Wiley-VCH.
• https://www.separations.us.tosohbioscience.com
• Lucy, C. A. (2003). "Evolution of ion-exchange: from Moses to the Manhattan
Project to Modern Times". Journal of chromatography. A. 1000 (1–2): 711–24.
• Ion Exchange Chromatography Principles and Methods. General Electric Company.
2004. pp. 11–20
• Jungbauer, Alois; Hahn, Rainer (2009). "Chapter 22 Ion-Exchange
Chromatography". Guide to Protein Purification, 2nd Edition. Methods in
Enzymology. 463. pp. 349–371.
• Jenke, D. (2011). "Application of Ion Chromatography in Pharmaceutical and Drug
Analysis". Journal of Chromatographic Science. 49 (7): 524–39.
• Gjerde, Douglas T.; Fritz, James S. (2000). Ion Chromatography. Weinheim: Wiley-
VCH