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Ion Exchange Chromatography

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20 views

Ion Exchange Chromatography

Uploaded by

mailcraag
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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ION EXCHANGE CHROMATOGRAPHY

Ion exchange chromatography is a process that allows the separation of ions and
polar molecules based on their affinity to ion exchangers.
The principle of separation is thus by reversible exchange of ions between the
target ions present in the sample solution to the ions present on ion-exchangers.
Ion exchange chromatography is most often performed in the form of
column chromatography.

Working Principle of ion exchange chromatography


This form of chromatography relies on the attraction between oppositely
charged stationary phase, known as an ion-exchanger, and analyte.
The ion exchangers basically contain charged groups covalently linked to the
surface of an insoluble matrix.
The charged groups of the matrix can be positively or negatively charged.
When suspended in an aqueous solution, the charged groups of the matrix will
be surrounded by ions of the opposite charge.
In this “ion cloud”, ions can be reversibly exchanged without changing the
nature and the properties of the matrix.

Two types of exchangers i.e., cationic and anionic exchangers can be used.
1. Cationic exchangers possess negatively charged group, and these will attract
positively charged cations. These exchangers are also called “Acidic ion
exchange” materials, because their negative charges result from the ionization
of acidic group.
Most common active sites for Cation exchange resin are the Sulfonic acid group
(a strong acid) and the carboxylic acid group (a weak acid)

2. Anionic exchangers have positively charged groups that will attract


negatively charged anions. These are also called “Basic ion exchange”
materials.
Anionic exchanger contains strongly basic tertiary amino group or weak basic
primary amino groups.

INSTRUMENTATION OF ION EXCHANGE CHROMATOGRAPHY


Typical Ion exchange chromatography instrumentation includes: pump, injector,
column, detector and recorder or data system.
1. Pump
The pump is considered to be one of the most important components in the
system which has to provide a continuous constant flow of the eluent through
the injector, column, and detector.

2. Injector
Sample introduction can be accomplished in various ways. The simplest method
is to use an injection valve. Liquid samples may be injected directly and solid
samples need only to be dissolved in an appropriate solvent. Injectors should
provide the possibility of injecting the liquid sample within the range of 0.1 to
100 ml of volume with high reproducibility and under high pressure (up to the
4000 psi).

3. Columns
Depending on its ultimate use and area of application, the column material may
be stainless steel, titanium, glass, etc. The column can vary in diameter from
about 2mm to 5 cm and in length from 3 cm to 50 cm depending on whether it
is to be used for normal analytical purposes, microanalysis, high speed analyses
or preparative work.
Guard column is placed anterior to the separating column. This serves as a
protective factor that prolongs the life and usefulness of the separation column.
They are dependable columns designed to filter or remove particles that clog the
separation column.

4. Detectors
Electrical conductivity detector is commonly use.

5. Data system
In routine analysis, where no automation is needed, a pre-programmed
computing integrator may be sufficient. For higher control levels, a more
intelligent device is necessary, such as a data station or minicomputer.
ION EXCHANGE PROCESS IS PERFORMED IN FOLLOWING
STEPS:

The first step (assurance of resin homogeneity)


It 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.

The second step (adsorption of sample)


It is the sample application and adsorption step, 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.

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.

The fourth and fifth steps (regeneration)


These steps 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.
Applications of ion exchange chromatography
• An important use of ion-exchange chromatography is in the routine
analysis of amino acid mixtures.
• The 20 principal amino acids from blood serum or from the hydrolysis of
proteins are separated and used in clinical diagnosis.
• This is most effective method for water purification. Complete
deionization of water is performed by exchanging solute cations for
hydrogen ions and solute anions for hydroxyl ions. This is called
softening of water.
• Separation of similar ions like lanthanides.
• Purification of Proteins

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