3. Introduction
Ion exchange chromatography (IEC) is a versatile and generic
tool for protein and plasmid separation.
Used in research, analysis and process-scale purification of
proteins.
In ion exchange chromatography , retention is based on the
attraction between the solute ions and charged sites bound to
stationary phase.
Columns used for ion exchanger are characterized by the
presence of charged groups covalently attached to the stationary.
Anion exchanger contain bound +ve groups, where as cation
exchanger contain bound –ve groups.
4. Apparatus
1) Column material and dimensions:-
Material:- Glass(laboratories) , High quality stainless steel or
polymers (industries).
Dimensions:- length: diameter ratio of 20:1 to 100:1
2) Type of ion exchange resin:-
a) According to the source:- Natural: Cation- Zeolytes, Clay
Anion- Dolomite
Synthetic:- Inorganic and organic resins
b) According to the chemical nature:-
Strong cation exchange resin (Sulfonic acid group).
Weak cation exchange resin (Methacrylate polymer).
Strong anion exchange resin (Quarternary ammonium).
Weak anion exchange resin (Methyl amino group).
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C) According to the structure:-
a) Pellicular type with ion exchange film:-
Particle size of 30-40 µ with 1-2 µ film thickness.
Ion exchange efficiency is 0.01-0.1 meq/g of ion exchange
resin.
b) Porous resin coated with exchanger beads:-
Particle size of 5-10 µ
Particle size of 0.5-2 meq/g of ion exchange resin
c) Macroreticular resin beads:-
Not highly efficient.
Very low exchange capacity.
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d) Surface sulfonated and bonded electrostatically with anion
exchange:-
Less efficient and low exchange capacity
Ion exchange capacity is 0.02 meq/g.
3) Stationary phase:-
It is composed of two structural elements; the
Charged groups which are involved in the exchanger process and
the matrix on which the charged groups are fixed.
Sever matrix materials are- Cellulose, Silica, polyacrylamide,
Acrylate co-polymer, Coated silica.
4) Mobile phase:-
Generally , eluents which consist of an aqueous solution of a
suitable salt or mixtures of the salt with a small percentage of an
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organic solvent are used in which most of the ionic compounds are
dissolved,
There are some eluents additives which have been used in ion
exchange chromatography- EDTA, Polyols, Glycerol, Glucose,
Detergents, Lipids, Organic solvents, Urea.
5) Buffers:-
In ion exchange chromatography, pH value is an important
parameter for separation and can be controlled by means of
buffer substances.
For Cation exchange chromatography- Citric acid. Lactic acid,
Acetic acid, Formic acid.
For Anion exchange chromatography- Piperazine, N-Methyl
Piperazine, Triethanolamine, Ethanolamine
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6) Sample preparation:-
For sample preparation ,the sample must be soluble in
eluents identically be dissolved in the mobile phase itself.
To protect the column from possible damage, samples are
filtered before use to remove particulates.
7) Packing of the column:-
Wet packing method is the ideal technique of column packing
in ion exchange chromatography.
Slurry is prepared by mixing silica (10-20g) and least polar
solvent in a beaker and poured in the column.
When the packing is complete the eluents is allowed to pass
through column for certain time.
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8) Development of the chromatogram and elution:-
After introduction of the sample, development of the
chromatogram is done by using different mobile phase.
There are two elution techniques- Isocratic and Gradient
elution.
9) Analysis of the elute:-
Different fraction collected with respect to volume or time is
analyzed for their contents by several methods.
Spectrophotometric method.
Polarographic method.
Conductometric method.
Radiochemical method.
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10) Regeneration of the ion exchange resin:-
Regeneration refers to the replacement of exchangeable
cations or anions present in the original resin.
Regeneration of cation exchange resin is done by charging
the column with strong acid like hydrochloric acid.
Regeneration of anion exchange resin is done by using
strong alkali like sodium hydroxide or potassium hydroxide.
11. Principle
The principle involved in the chromatography is the attraction
between oppositely charged particles.
Many biological material such as amino acids and protein, have
ionisable groups and they carry a net positive or negative charge
can be utilized in separating mixtures of such compounds.
The net charge of such compounds is dependent on their pka and
pH of the solution.
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1) Pump:- The IC 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 IC injector,
column, and detector
Pumps types:-
a) Reciprocating piston pumps.
b) Dual piston pumps.
2) Injector:-Sample introduction can be accomplished in
various ways. The simplest method is to use an injection
valve. In more sophisticated LC, automatic sampling devices
are incorporated where sample introduction is done with the
help of auto-samplers and microprocessors. 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).
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3) Columns:- Glass(laboratories) , High quality stainless steel
or polymers (industries).
4) Suppressor:- The suppressor reduces the background
conductivity of the chemicals used to elute samples from the
ion-exchange column which improves the conductivity
measurement of the ions being tested.
5) Detectors:- An ideal detector should have the following
properties:
low drift and noise level.
high sensitivity.
fast response.
wide linear dynamic range.
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Types of detector:-
a) Electrical conductivity detector.
b) Amperometric detector.
c) Fluorimrtric detector.
d) Photometric detector.
6) Data system:-
The main goal in using electronic data systems is to increase
analysis accuracy and precision, while reducing operator
attention. In routine analysis, where no automation (in terms
of data management or process control) 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.
20. Parameters
1) Rate of ion exchange process:-
The rate of ion exchange process depends on the rate of
diffusion ,which is the slowest state in ion exchange process.
2) Flow rate:-
Due to differences in rates of exchange and the fact that they
may vary significantly for different kinds of separation, flow rates
are to be controlled (0.5-5 ml/min).
3) Particle size:-
Large surface area and small particles will increase the rate of
ion exchange process.
4) Porosity:-
High porosity offers a large surface area covered by charged
groups and so provides a high binding capacity.
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5) Total capacity:-
Total capacity of a resin is determined by taking a weighed
sample of resin, placing it in a column and passing through a
solution of Kcl through the column in excess.
6) Swelling:-
Swelling is due to the tendency of particles to hydrate and
electrostatic repulsion of fixed ions with like charges.
Swelling is continued until an equilibrium is reached between the
osmotic pressure in the system and the opposing elastic force of
the flexible hydrocarbon chains.
22. Factors affecting resolution
Nature of exchanging ions.
Nature of ion exchange resin.
Chemical variables.
Physical variables.
Ion exchange constant.
Surface area.
Temperature.
Composition of cation exchange resin
Length of the column
23. Applications
Separation of similar ion from one another .
Removal of interfering radicals.
Softening of hard water.
Complete demineralization of water.
Separation of lanthanides.
Separation of actinides.
Separation of amino acids.
Separation of sugars.
24. References
Jungbauer, Alois, and Rainer Hahn. "Ion-exchange
chromatography." Methods in enzymology. Vol. 463.
Academic Press, 2009. 349-371.
Selkirk, Chris. "Ion-exchange chromatography." Protein
Purification Protocols. Humana Press, 2004. 125-131.
Instrumental methods of chemical analysis ,
Gurudeep.R.Chatwal , Sham.k.Anand pg no: 2.662-2.672.
Pharmaceutical analysis-II “ instrumental methods “ by P.C
Kamboj pg: 189-198.
Moustafa, Yasser M., and Rania E. Morsi. "Ion exchange
chromatography-An overview." Column Chromatography.
InTech, 2013.
