Chrom-Lect 4-Ion Exch

Chromatographic Methods
of Analysis
Section - 4 : Ion Exchange Chrom.
Prof. Tarek A. Fayed

Ion Exchange Chromatography (IEC)
 In this type of chromatography, the solid stationary
phase )organic resin) is containing covalently bonded
anions or cations onto it. Solute ions of the opposite
charge in the mobile liquid phase (electrolyte) are
attracted to the resin by electrostatic forces, and then
exchanged
 Thus solutes are separated due to the differences in the
type and magnitude of their ionic charges. The
separation is due to exchange of ions in the sample with
the labile ions onto the stationary phase (exchange
resin).
Electrolyte
+ Resin
Structure of Ion-Exchangers
Cation exchangers: -SO3-, -CO2- the labile ion is H+ or Na+

Anion exchangers: -NR3+ the labile ion is OH- or Cl-
Shape of Ion Exchangers
4
Separation in ion exchange chromatography depends
upon the reversible adsorption of charged solute
molecules to the immobilized ion exchange groups of
opposite charge onto the resin beads.
Properties of Resin
Any ion exchanger
should be;
 Chemically and
mechanically stable.
 Chemically inert.
 Have the same degree
of cross-linking and
mesh size.
 Homogenous (one
type; either cationic or
anionic)
Anion exchangers are positively charged exchangers and have
negatively charged counter labile-ions (anions) available for exchange.
Cation exchangers are negatively charged exchangers and have
positively charged counter labile-ions (cations) available for exchange.
Ion exchange process is performed in five main
steps:
 1. The first step (assurance of resin homogeneity)
is an equilibration in which the ion exchanger is brought
to a starting state, which allows the binding of the
desired solute molecules. The exchanger groups are
associated at this time with exchangeable counter labile-
ions (usually simple anions or cations, such as chloride
or sodium).
 2. The second step (adsorption of sample)

Is the sample application and adsorption, in which solute
molecules carrying the appropriate charge displace
counter-ions and bind reversibly to the resin surface.
Unbound substances can be washed out from the
exchanger bead using eluent (buffer or electrolyte)
 3. The third step (desorption and elution of
components)
The substances are removed from the column
by changing to elution conditions for ionic
bonding of the solute molecules. This normally
involves increasing the ionic strength of the
eluting buffer or changing its pH.
 4. The fourth and fifth steps (regeneration)

Are the removal of substances from the column
that are not eluted under the previous
experimental conditions and re-equilibration at
the starting conditions for the next purification.
Exchange constant or selectivity coefficient (K)
 xRN(CH3)3+OH- + Ax- (RN(CH3)3)x+Ax- + xOH-

Stationary Mobile Stationary Mobile
 xRSO3H + Mx+ (RSO3-)xMx+ + xH+

Stationary Mobile Stationary Mobile
For the above equilibria;
K = [R-M+][H+]/[R-H+][M+]
Factors affecting ion exchange selectivity (K)
 Three major factors are affecting ion exchange
selectivity;
1. Nature and properties of ion exchange resins
 Cross linking and swelling are important factors, when
more cross linking agent is present, the resin becomes
more rigid and swells less (has small pore size). This
makes separations of ions of different sizes more
difficult as they can not pass through the pores present
and it becomes selective to ions of different (smaller)
sizes.
 The nature of resin whether cationic or anionic
exchanger, which determines strongly its selectivity.
Cationic resin is selective for cations and vice versa.
 Also, the resin capacity (number of me-equivalents of
replaceable ions per gram of dry resin) is important.
How it could be determined?
2.Nature of exchanging ions in the sample
a. Valence of ions:
 At low concentrations, the extent of exchanges
increases with increase in valence; Ions with higher
charge is more selective;
Ce4+ < Al3+ > Ba2+ > Ti+
PO33- < SO42- > NO3-
b. Size of ions:
 For similarly charged ions; the exchange selectivity
increases with decrease in the size of hydrated ions;
Li+ < H+ < Na+ < NH4+ < K+ < Rb+ < Cs+ < Ag+
and; Mg2+ < Ca2+ < Ba2+ > Sr2+
SO42- > Cr2O42- > I- > NO3- > Br - > Cl- > HCO2- > OH- > F-
c. Polarizability:
Highly polarizable ions are more selective. Exchange is
preferred for greater polarizable ions, as; I- < Br- < Cl - < F-
d. Concentration of solutions:
In dilute solutions poly-valent ions are generally absorbed
preferentially.
3.Nature of mobile phase and pH
 The presence of other ions that compete with the sample
for binding to the ion exchanger (using of electrolyte).
 The pH of the solution which influences the net charge of
the sample (as in case of amino acids).
Applications of Ion Exchange Chromatography
1- Clinical applications:
As in separation of amino
acids and proteins.
Ion exchange chromatography
is a principle technique for
analyses of the amino acids and
proteins.
Proteins move with rates determined
by their net charge at the used pH of
the eluent. With cation exchangers,
proteins with more negative charge move faster
and eluted earlier, then less negative and less positive.
Finally, the more positive ones.
For separation of amino acids mixture in
protein hydrolysate using cation exchanger;
 Amino acids have functional groups that can carry
both positive or negative charges (amphoteric)
depending on the pH or the ionic concentration of the
mobile phase.
 Ion exchange chromatography separates amino acids
according to their net charge and iso-electric points (PI)
which is dependent on the pH of the mobile phase.
 For example; if a mixture of aspartic, glycine and
arginine has been introduced into cationic resin in a
strong acidic buffer, then on gradient elution using
increasing pH, results in sequential elution of more
acidic aspartic, followed by neutral glycine and finally
the more basic arginine .
2- Separation of similar ions like lanthanides
Lanthanides have
very similar
properties and can
not be separated by
usual techniques,
but can be separated
by ion exchange
chromatography.
This depends on the
Acidity (ionic
strength) of the
medium.
3- Removal of interfering ions
For estimation of Ca+2, Ba+2 ions by the oxalate or sulphate
method in quantitative analysis, phosphate ions are found to
interfere and can be removed by passing the solution through
H2SO4.
4- Pre-concentration of solutions
By passing much water over a resin and then elute with a
high concentration of acid. Cation exchangers trap cations.
It is important for trace analysis, where solubility [s] is extremely
low . It is important for environmental problem.
5- Water deionization and softening
Removal of cations by cation exchangers and anion exchangers
for anion removal.
ex: Removing of toxic lead ions from seawater
Schematic representation of ion exchange Chromatograph, as HPLC
The chromatogram of some anions separated by using
anion exchanger.

Chrom-Lect 4-Ion Exch

Uploaded by

Copyright:

Available Formats

Chrom-Lect 4-Ion Exch

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Chrom-Lect 4-Ion Exch

Uploaded by

Copyright:

Available Formats

Chromatographic Methods

Prof. Tarek A. Fayed

Cation exchangers: -SO3-, -CO2- the labile ion is H+ or Na+

 2. The second step (adsorption of sample)

 4. The fourth and fifth steps (regeneration)

 xRN(CH3)3+OH- + Ax- (RN(CH3)3)x+Ax- + xOH-

 xRSO3H + Mx+ (RSO3-)xMx+ + xH+

For the above equilibria;

You might also like