Definition
• Ion-exchange
chromatography
• (or ion chromatography) is a
process that allows the
separation of ions and polar
molecules based on the charge
properties of the molecules.
2
Ion Chromatography (IC) was introduced in 1975 by Small, Stevens, and
Bauman as a new analytical method.
In 1979, Fritz et al. described an alternative separation and detection
scheme for inorganic anions, in which the separator column is directly
coupled to the conductivity cell.
At the end of the 1970s, ion chromatographic techniques were used to
analyze organic ions for the first time.
The 1980s witnessed the development of high efficiency separator columns
with particle diameters between 5 µm and 8 µm, which resulted in a
significant reduction of analysis time.
Since the beginning of the 1990s column development has aimed to provide
stationary phases with special selectivity.
1. Ion-Exchange Chromatography (HPIC)
(High Performance Ion Chromatography)
2. Ion-Exclusion Chromatography (HPICE)
(High Performance Ion Chromatography Exclusion)
3. Ion-Pair Chromatography (MPIC)
(Mobile Phase Ion Chromatography)
This separation method is based on ion-exchange processes occurring
between the mobile phase and ion-exchange groups bonded to the support
material.
Ion-exchange chromatography is used for the separation of both inorganic
and organic anions and cations.
Ion-exclusion chromatography is particularly useful for the separation of weak
inorganic and organic acids from completely dissociated acids which elute as
one peak within the void volume of the column. In combination with suitable
detection systems, this separation method is also useful for determining amino
acids, aldehydes, and alcohols.
The dominating separation mechanism in ion-pair chromatography is
adsorption. The stationary phase consists of a neutral porous divinylbenzene
resin of low polarity and high specific surface area.
Ion-pair chromatography is particularly suited for the separation of surfaceactive anions and cations, sulfur compounds, amines, and transition metal
complexes.
An ion exchanger consists of an insoluble matrix to which charged
groups have been covalently bound. The charged groups are
associated with mobile counter-ions. These counter-ions can be
reversibly exchanged with other ions of the same charge without
altering the matrix.
1-Positively charged exchangers have negatively charged counter-ions
(anions) available for exchange and are called anion exchangers.
2-Negatively charged exchangers have positively charged counter-ions
(cations) and are termed cation exchangers.
)DEAE-cellulose( ‫ دی اتیل آمینو سلولز‬Anion exchanger ‫( پلی ساکاریدی‬1
)CM. Cellulose( ‫ کربوکس ی متیل سلولز‬Cation exchanger
‫ دی وینیل بنزن‬،‫ وینیل بنزن‬: ‫( سنتتیک‬1
An almost unlimited variety of resins with different compositions and
degrees
of cross linking can be prepared. The resins consist of an elastic threedimensional network of hydrocarbons which carry fixed ionic groups
In a cation exchanger, the matrix carries ionic groups like
− SO3−, − COO−, − PO3
and in an anion exchanger, it carries groups such as
−NH3+, >NH2 , > N+
Anion
exchanger
• Aminoethyl (AE-)
• Diethylaminoethyl
(DEAE-)
• Quaternary
aminoethyl (QAE-)
Cation exchanger
•
•
Carboxymethyl cellulose
(CM-cellulose)
Sulphopropyl (SP-)
amphoteric
exchangers
snake- cage
Polyelectrolytes
• Cation exchange chromatography retains
positively charged cations because the
stationary phase displays a negatively
charged functional group
- +
+ -
R-X C +M B
_
+
+
-
R-X M + C + B
• Anion exchange chromatography retains
anions using positively charged functional
group:
+ +
+ + _
R-X A +M B
R-X B + M + A
Preparation of resin
pH of buffers
Buffer selection
Elution
PH
change in ionic strength
Procedure
Dr Gihan Gawish
Effect of pH in the separation of
proteins
• By adjusting the pH or the ionic concentration of
the mobile phase, various protein molecules can
be separated.
• For example, if a protein has a net positive
charge at pH 7.0, then it will bind to a column of
negatively-charged beads, whereas a
negatively charged protein would not.
Effect of pH in the separation of
proteins
• Proteins are charged molecules. At
specific pH, it can exist in anionic (-),
cationic (+) or zwitterion (no net charge)
stage.
cationic
pH =pI
pH increase
*pI isoelectric point
anionic
Choosing your ion-exchanger: know
your proteins
1. Stability of proteins
 stable below pI value, use cation-exchanger
 stable above pI value, use anion-exchanger
2. Molecular size of proteins
 <10,000 MW, use matrix of small pore size
 10,000-100,000 MW, use Sepharose equivalent
grade
Detection In Ion Chromatography
1. Conductivity detection
2. Electrochemical (amperometric or coulometric) detection
3. Potentiometric detection
4. Spectroscopic detection
5. Post-column reaction detection.
Detection by post-column reaction (PCR) involves the chemical reaction of the
solutes as they elute from the column on the fly, prior to their introduction to the
detector.
• Sensitivity
• Linearity
• Resolution (detector cell volume)
• Noise (detection limit(
• Sample loading capacity does not decrease at high or low pH values due
to loss of charge from the ion exchanger .
• A very simple mechanism of interaction exists between the ion exchanger
and the solute.
• Ion exchange experiments are more controllable since the charge
characteristics of the media do not change with changes in pH. This makes
strong exchangers ideal for working with data derived from electrophoretic
titration curves.
Ion-exchange chromatography separates proteins by charge under near
physiological and non-denaturing conditions.
its widespread applicability, its high resolving power
. Speed
• Sensitivity
• Selectivity
• Simultaneous detection
• Stability of the separator columns
Enzyme
The recovery of creatine kinase in this separation
was 89%.
isoenzyme
Immunoglobulins