this slide contains all the basic about the topic ion exchange chromatography which contains all important information about topic in very easy language. it will be helpful for BSc, pharmacy and biomedical student.

1. ION-EXCHANGE
CHROMATOGRAPHY
Presented by – Rakshit Bamola M Pharm (pharmaceutics)
Under the supervision of – Dr Amita Joshi Rana

2. Contents
• Introduction
• Principle
• Methodology
• Instrumentation
• Advantages & disadvantages
• Applications
• Reference

3. Introduction
• Chromatography – separation of sample component after their
distribution between two phases (stationary phase & mobile phase).
• In ion exchange chromatography (IEC) separation takes place on
the basis of net charge, present on the sample component.
• This method is used for separation of protein, nucleotide and amino
acids or purification of their mixture based on their net charge.
• Segregation of molecules based on ion displacement theory. This
means a more potent ion displaces a weaker ion.
• This technique has been developing since 19th century which was
firstly used for purifying the drinking water.

4. Principle
• Protein vary according to their net charge at neutral ph.
• Proteins made up of amino acids they have charged side chains, for
example- positive side chain - lysine, arginine & negative side chains -
aspartate, glutamate.
• If we want to produce charge onto the protein then we can very there ph.
according to their Isoelectric point and we can get desired charge on to
them.
• Column in IEC filled with charged resins & they are of 2 type – 1) Cation
exchange resin, 2) Anion exchange resin.
• On the basis of counter ion which exchanges with sample molecule, resins
classified as cation and anion exchange resin.

5. Principle…
Pictorial representation of cation and anion exchange resin

6. Principle…
• To understand how IEC work, we assume we have a mixture of 3 protein
namely A, B & C and we have to separate them.
• ‘Protein A’ contain highly negative charge ‘protein B’ low negative charge
and ‘protein C’ contain positive charge’.
• We have filled column with a anion exchange resin.
• When sample introduced into the column with the help of mobile phase,
‘protein A’ & ‘protein B’ replaces the counter ion of resin & protein c
repelled due to presence of same charge.
• ‘Protein A’ will bind to the resin strongly due to higher negative charge,
‘protein B’ will also binds to the resin but in lesser extent compare to
‘protein A’, due to its lesser negative charge compare to ‘protein A’.

7. Principle…
Pictorial representation of ion replacement in anion exchange resin

8. Methodology
The process is of 4 steps -
Equilibration Loading
Washing Elution

9. Methodology…
1) Equilibration
• In this step we attach the appropriate counter ions to the previously
filled ion exchange resin.
• Resin present in column have a charge then the appropriate counter-
ion (usually Na+, Cl- , H+, OH-) solution introduced into the column.
• Due to electrostatic forces the counter-ions attached to the resin.
• Buffer solution which will be used as mobile phase ran through
column to wash out unattached ions, we run mobile phase until we get
straight line.
• When base line comes it means column/instrument is ready for next
step.

10. Methodology…
2) Loading
• Buffer solution (mobile phase) ph. maintained according to the
demand of the analyte ion
• Sample solution simply injected or mixed with the buffer solution and
loaded to the column.
• Competition between counter ion and analyte ion takes place for
binding into the resin.
• Exchange of ion takes place

11. Methodology…
3) Washing
• Unbound molecules like unbound analyte and replaced ions washout
in this steps with the help of starting buffer solution
• If sample have some protein/ nucleic acid which have same charge like
counter ion may elute out during this stage
• Graph obtained during washing looks like-

12. Methodology…
4) Elution
• This is the most important steps amongst all, because separation of ion
takes place in this step.
• since different substances have different degrees of interaction with
the ion exchanger due to differences in their charges, charge densities
and distribution of charge on their surfaces.
• These interactions can be controlled by varying conditions such as
ionic strength and ph. Of the buffer solution.
• Elution can carried out by 2 methods – a) Salt gradient method & b)
ph. gradient method.

13. Methodology…
4) Elution…
a) Salt gradient method
• In this method we increase the same ion salt concentration gradually
which replaces the analyte ion, same as the analyte ion replaces the
counter ion
• Then after gradual increase of salt all analyte separated according to
their bond strength.
• Resin which degrades due to ph. change this method suited best for
them.

14. Methodology…
4) Elution…
b) ph. gradient method
• Every protein have specific Isoelectric point (PI) (where protein
become zwitterion) which can be calculated by Henderson-
Hasselbach equation or simply by some computer based method.
• If we use buffer which have same ph. as PI of the analyte, then analyte
become neutral, hence binding with resin disrupted, and by gradually
increasing buffer ph we can elute out all the analyte present in the
sample one by one.

15. Instrumentation
• Mobile phase
• Pump
• Sample injector
• Column
• Stationary phase
• Detector
• Read out system- Graph

16. Instrumentation…
Pictorial representation of basic instrumentation of ion exchange chromatography

17. Instrumentation…
Mobile phase
• Different strengths of acids, alkalis and buffers are used as eluting
solvent
• Mobile phase selection according to their ph. is important it should be
selected according to the nature of sample and the resin we are using.
• For cation exchange chromatography, the elution buffer pH is
maintained in the range 4-7 and for anion exchange in the range 7-11
• At very low or very high pH values, the analyte, especially proteins,
may bind strongly to the stationary phase, requiring high salt
concentrations for their elution.

18. Instrumentation…
Mobile phase…
• Some proteins may precipitate or lose their activity at high pH values
and high salt concentrations so it should avoided.
• Typical buffers used as mobile phases for cation exchange
chromatography include Formate, Acetate, MES, Phosphate or Tris.
• While buffers that are used as mobile phases for anion exchange
chromatography include Piperazine and Diethylamine (11).

19. Instrumentation…
Pump
• A high-pressure pump is considered as one of the main parts of the Ion
exchange chromatography system which delivers a constant flow of
the eluent.
Sample Injector
• Amongst the various ways for sample introduction into the eluent
stream and onto the column, the most straightforward method being
the use of an injection valve.
• Other HPLC injection system can also used in IEC.

20. Instrumentation…
Column
• Columns being utilized in the laboratories are built from glass whereas
the columns utilized in industries are either fabricated from high-
quality stainless steel or polymer.
• Separation improved by increasing length of the column but length can
not be increased beyond a critical length
• Generally ratio of 10:1 or 100:1 of height to diameter is better for
efficient separation

21. Instrumentation…
Stationary phase
• Column packed with the ion exchange resins.
• Anion or cation exchange resin – some proteins are stable only
above or below their pI. For these proteins, stability dictates resin
choice; if, for example, a protein is stable only above its pI, an anion
exchange resin should be chosen. When protein stability is not of
concern, either an anion or cation exchanger can be used.
• Resins are classified variously some of their classification are given
here
1. According to source- natural, synthetic organic & synthetic
inorganic

22. Instrumentation…
Stationary phase…
1) According to source…
Natural organic ion exchanger -
• coal, paper, cotton etc. can be converted into cation exchange by
reaction of sulphonation or phosphorylation
• These materials are less uniform in structure and readily affected by
other chemicals
• Mostly Zeolite & Clay used as cation & Dolomite used as anion
exchange resin

23. Instrumentation…
Stationary phase…
1) According to source…
Synthetic organic ion exchangers –
• Made up of Synthetic polymer network and reacting them with various
functional groups
• Nature of functional group determine whether it is cation or anion
exchanger
• Number and type of functional group determine whether it is strong or weak
ion exchanger.
• E.g. Polystyrene (exchangeable functional group) + divenylbenzene (cross
linking- stability

24. Instrumentation…
Stationary phase…
According to source…
Synthetic inorganic ion exchanger –
• Have three dimensional frame work structure with channels and
interconnecting cavities
• E.g. Alumino-silicate, Titanium dioxide, Thorium dioxide, Zirconium
dioxide, Phosphates etc.

25. Instrumentation…
Stationary phase…
2) According to chemical structure –
Weak anion exchanger - Diethyl amino ethyl compound
Strong anion exchanger– Quaternary ammonium compound
Weak cation exchanger - Carboxyl methyl compound
Strong cation exchanger - Sulphonic acid
Cellulose & dextran ion exchangers which are polymer of glucose
have larger pore size and low electron density but they are much softer
than resins so they are classified sometimes as ion exchange gels

26. Instrumentation…
Stationary phase…
Essential property needed in ion exchange resin -
• Minimum particle size
• insoluble in common solvents
• Optimum Cross linkagent
• Chemical stability
• Regeneration capability
• Good ion exchange capacity (m.eq/gm)
• Optimum flow rate

27. Instrumentation…
detector
• They estimate the analyte peaks as soon as the eluent comes out of the
column
• Conductivity detectors used mostly which are best suited for IEC, they are
universal for charged species (more ions - more conductivity)
• They respond sensitively and permit quantitative determination of ions on
the basis of chromatographic peak areas.
• High electrolyte concentration of eluent effected the sensitivity of this
detector to overcome this “suppressor” are attached to the column.
• Suppressor wall saturated with opposite ions which attract the extra ions of
eluent and neutralize them due to which clear spectra obtained

28. Instrumentation…
Read out system - Graph

29. Advantage and Disadvantage
Advantages Disadvantages
Most efficient methods for the
separation of charged particles
Separation is possible of charged
molecules only
Can be used for almost any kind
of charged molecule including
large proteins, small nucleotides
and amino acids
Buffer requirement
It offers high selectivity: it can
resolve molecule with high
difference in charge
Efficiency of the column is less

30. Application
1. Separation of similar charged ions.
2. Demineralization of water.
3. Softening of hard water.
4. Separation of sugars.
5. Purification and recovery of pharmaceuticals.
6. Biochemical separations

31. Reference
• 1. D. A. Skoog, F. J. Holler and S. R. Crouch, Principles of Instrumental Analysis
• 12. J. W. Robinson, E. M. S. Frame and G. M. Frame II, Undergraduate
Instrumental Analysis
• https://www.bio-rad.com/en-in/applications-technologies/ion-exchange-
chromatography?ID=MWHAY9ESH
• https://bitesizebio.com/31744/basics-ion-exchange-chromatography/
• https://www.pharmatutor.org/pdf_download/pdf/Vol.%205,%20Issue%202,%20Fe
bruary%202017,%20PharmaTutor,%20Paper-5.pdf
• https://www.sciencequeue.com/ion-exchange-chromatography/
• B.D. Shashikant, Rageeb, P.A Salunke, S.S. patil, Modern Pharmaceutical
Analytical Techniques.