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assignment ion exchange chromatography
1. ION EXCHANGE CHROMATOGRAPHY
Introduction :
Ion exchange chromatography is used to remove ions of one type from a mixture and
replace them by ions of another type . The column is packed with porous beads of a resin
that will exchange either cations or anions. There is one type of ion on the surface of the
resin and these are released when other ions are bound in their place – e.g. a basic anion
exchange resin might remove nitrate ions (NO3–) from a solution and replace them with
hydroxide ions (OH–).
Principles:
• Separation is based on the attraction between oppositely charged particles.
• Net charge exhibited by compound is dependent on their pKa and pH of the solution
in accordance with HENDERSON HASSELBACH EQUATION.
• Ion exchangers special type of polyelectrolyte and consist of to which are bonded a
large number of electrically charged group.
• Classification: Ion exchangers
Based on the inorganic group
Based on the nature of the source
Based on the structure
Anion exchangers:
Anion exchangers contain bound positive groups, where as cation exchangers contain
bound negative groups.
Cation exchangers:
Cationic exchangers are useful for separation of cations such as protonated bases and
anion exchange columns are used for anions or acidic samples.
Fig: Ion exchange chromatography
2. Resin:
• Resins are amorphous particles of organic materials
• Polystyrene resins for ion exchange are made by co-polymerization of styrene and
divinyl benzene.
• Divinyl benzene content is varied from 1 to 16 percent to increase the extent of cross
linking.
• Benzene groups are modified to produce cation exchange resin and anion exchange
resin.
Column & media preparation:
Equilibrate column with 5–10 column volumes of start buffer or until the baseline,
eluent pH and conductivity are stable.
Using prepacked columns is highly recommended to ensure the best performance
and reproducible results. An evenly packed column ensures that component peaks
are not unnecessarily broadened as sample passes down the column so that the best
resolution can be achieved.
Sample applications:
Desalt the sample and transfer to the buffer
Adjust the sample to the chosen starting pH and ionic strength and apply to the
column.
Sample volume should be based on the capacity of ion exchange resins
For protein samples maximum concentration of about 50- 70mg/ml can be used.
Sample loading:
Apply samples directly to the column via a chromatography system, a peristaltic
pump or a syringe.
The choice of equipment depends largely on the sample volume, the size of
column, the type of IEX medium and the requirements for accuracy in gradient
elution.
Ensure that the top of the column bed is not disturbed during sample application
Do not change buffer conditions until all unbound material has been washed
through the column (monitored by UV absorbance) and until UV and conductivity
values have returned.
Re Equilibration:
Wash with 5 column volumes of 1 M NaCl (100%B) to elute any remaining
ionically-bound material. Include a wash step at the end of every run in order to
remove any molecules that are still bound to the medium. Monitor UV absorbance
so that the wash step can be shortened or prolonged, as necessary.
3. Re-equilibrate with 5–10 column volumes of start buffer or until eluent pH and
conductivity reach the required values. A re-equilibration step after washing returns
the column to start conditions before applying further samples. Whenever possible,
monitor pH and conductivity to check when start conditions have been reached. The
re-equilibration step can then be shortened or prolonged as necessary.
Analysis of eluent:
Spectrophotometric method.
Flame photometry
Conductometric methods
Radio isotope method
Advantages:
• Detectability: useful for the detection of many in-organic salts and also for the
detection of organic ions with poor UV absorptivity like alkyl amines or sulfonates.
• Preparative separations: usually preferred because of the availability of volatile
buffers . volatile buffers makes the removal of mobile phase easier.
• Useful to resolve very complex samples, i.e in the case of multi step separation
• Useful for separation of mixtures of biological origin, in organic salts and some
organo- metallic
Disadvantages:
• Column efficiency is less
• It is difficult to achieve control over selectivity and resolution
• Stability and reproducibility of the columns become questionable after repeated use.
Applications:
• Ion exchange chromatography is used to convert one salt to other.
• It is useful for pre concentration of trace components of a solution to obtain enough
for analysis.
• Ion exchange is used to prepare de-ionized water.
• Water polishing equipment used in many laboratories uses several ion exchange
cartridges.
4. Conclusion:
Ion exchange chromatography has many advantages. This method is widely
applicable to the analysis of a large number of molecules with high capacity. The
technique is easily transferred to the manufacturing scales with low cost. High levels
of purification of the desired molecule can be achieved by ion exchange step. Follow-
up of the nonsolvent extractable natural products can be realized by this technique.
References:
Instrumental analysis of chemical analysis by Gurudeep R.Chatwal,
Sham.K.Anand, Pg. 2.662- 2.672
Instrumental methods of chemical analysis by B.K.sharma, C-123 -150