ION EXCHANGE CHROMATOGRAPHY
By M.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
ION EXCHANGE CHROMATOGRAPHY
By M.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
This presentation contains all the topics related to column chromatography. That includes introduction, principle,apparatus, experimental aspects of column chromatography, application of column chromatography, advantage and disadvantage of column chromatography with reference.
Introduction to chromatography, Definition of Chromatography, Types of column chromatography, Theory of chromatography, Practical considerations in column chromatography , Factors affecting efficiency of a column, Applications.
This is a type of chromatography in which similar charged ions are separated by using ion exchange resin, that exchanges ions according to their relative affinities.
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
This presentation contains all the topics related to column chromatography. That includes introduction, principle,apparatus, experimental aspects of column chromatography, application of column chromatography, advantage and disadvantage of column chromatography with reference.
Introduction to chromatography, Definition of Chromatography, Types of column chromatography, Theory of chromatography, Practical considerations in column chromatography , Factors affecting efficiency of a column, Applications.
This is a type of chromatography in which similar charged ions are separated by using ion exchange resin, that exchanges ions according to their relative affinities.
Ion exchange chromatography works under the principle of reversible adsorption and this method involves the separation of ions by using different types of exchange resins based on the ions to be separated.
Chromatography, chromatography techniques,ion exchange chromatography, elution based chromatography,types of chromatography,types of resin,ideal characteristics of resin, physical properties of resin, factors affecting ion exchange process, application of resin, elution of resin, regeneration of resin, Equilibration in ion exchange chromatography, application of ion exchange chromatography
Download and play it my friends it contain VIDEO
The technique of ion exchange chromatography is based upon the interaction between charged solute molecules and oppositely charged moieties covalently linked to chromatographic matrix.
The reasons for its widespread success is its applicability, high resolving power, high capacity and simplicity of the technique.
Separation in ion exchange chromatography depends upon the reversible adsorption of charged solute molecules to immobilized ion exchange groups of opposite charge. Most experiments are performed by following : Video For Understanding Play It
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2. CONTENTS
INTRODUCTION OF IEC
PRINCIPLE OF SEPARATION
ION EXCHANGE RESINS (CLASSIFICATION
OF RESINS)
PRACTICAL REQUIREMENTS
FACTORS AFFECTING ION EXCHANGE
SEPARATION
APPLICATIONS
3. ION EXCHANGE CHROMATOGRAPHY
DEFINITION:
Ion exchange chromatography is a
process by which a mixture of similar
charged ions can be separated by using an
ion exchange resin which exchanges ions
according to their relative affinities.
4. INTRODUCTION
Ion exchange chromatography is a type of
adsorption chromatography.
There is a REVERSIBLE EXCHANGE OF
SIMILAR CHARGED IONS.
Mostly similar charged ions like cations and
anions can be conveniently separated by this
technique.
Many drugs and pharmaceutical agents are
weakly or strongly acidic or basic in nature.
Hence a mixture of similar charged substances
can also be separated into pure components.
5.
6. PRINCIPLE OF SEPERATION
This is by reversible exchange of ions between the ions
present in the solution and those present in the ion
exchange resin.
CATION EXCHANGE:
The separation of cations using cation exchange resin.
The cations to be separated are present in solution and
exchanges for similar ions present in cation exchange
resin, a solid matrix. the exchange can be represented
by the following equation:
X+ + R−K+ X+R− + K+ (solution)
(solution)
7. The cations retained by the solid matrix of ion
exchange resin can be eluted by using buffers of
different strength and hence separation of cations can
be effected.
ANION EXCHANGE:
Separation of anion using anion exchange resin can
be carried out. The anions to be separated are present
in solution and exchanges for similar ions present in
anion exchange resin, a solid matrix the exchange can
be represented by the following equation:
(solid)
X- + R+Cl- X-R+ + Cl- (anion
exchange)
(solution )
8. The anions retained by the solid matrix of ion
exchange resin can be eluted by using buffers of
different strength and hence separation of anions can
be effected.
9. CLASSIFICATION OF
ION EXCHANGE RESINS:
There are 6 types,
1. Source of the resin
2. Chemical nature
3. Functional group
4. Structural type
5. Physical properties
6. Chemical structure
Ion exchange resin should have following requirements
» It must be chemically stable.
» It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
» The swollen resin must be denser than water.
» It must contain sufficient no. of ion exchange
groups.
10. Divided in to two,
1. Natural
cation – Zeolytes, Clay, etc
Anion – Dolomite
2. Synthetic
In organic and Organic resins
1
11. 2. Synthetic
In organic and Organic resins
Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional
groups)
Divinyl benzene(Cross linking agent)-offers stability.
2
2. According to chemical nature:
4 types:
1. Strong cation exchange resin
2. weak cation exchange resin
3. Strong anion exchange resin
4. weak anion exchange resin
12. Strongly acidic cation exchanger ---sulphonic acid
groups attached to styrene and di vinyl benzene copolymer.
Weakly acidic cation exchanger---carboxylic acid groups
attached to acrylic and divinyl benzene co-polymer
Strongly basic anion exchanger-----quaternary
ammonium groups attached to styrene and divinyl benzene
co-polymer N+
Weakly basic anion exchanger-----poly alkyl amine
groups attached to styrene and divinyl benzene co-polymer
FUNCTIONAL GROUPS PRESENT IN DIFFERENT ION
EXCHANGE RESINS:
Strong cation exchange resin- SO3H
Weak cation exchange resin- COOH,OH,SH,PO3H2
Strong anion exchange resin- N+R3,NR2
Weak anion exchange resin- NHR,NH2
13. STRUCTURAL TYPE OF ION EXCHANGE
RESINS
sins:
It contains 4 types. They are,
1. Pellicular type with ion exchange film
2. Porous resin coated with exchanger beads.
3. Macro reticular resin bead.
4.Surface sulfonated and bonded electro
statically with anion exchanger
a) Pellicular type with ion exchange re
»30 - 40µ with 1-2µ film thickness
»Very low exchange capacity
» their ion exchange efficiency is 0.01- 0.1 meq/g
of ion exchange resin.
14. b) Porous resin coated with exchanger beads
» Size 5 - 10µ
» they are totally Porous & highly efficient.
» their exchange capacities are from 0.5- 2
meq/g of ion exchange resin
c) Macroreticular resin bead
» Not highly efficient & very low exchange
capacities.
15. d) Surface sulfonated and boned electrostatically with anion
exchanger:
» The particles are sulfonated and they are bonded
electrostatically with anion exchanger resin.
» They are less efficient and have low exchange capacity.
» their exchange capacity is 0.02meq/g of exchange
resin.
16. PRACTICAL REQUIREMENTS:
1. Column material and dimension
2. Type of ion exchange resin & the selection depend on
following properties
a.) type of ions
b.) nature of ions
c.) efficiency of resin
d.) Particle size
e.) Structural type
3. Packing of column
4. Mobile phase – acids, alkali, and buffers.
5. Development of chromatogram
6. Analysis of the elute
7. Regeneration of ion exchange resin
17. 1. Column material and dimensions:
Columns used in the laboratories are made up of
glass. In industries are made up of either high
quality stainless steel or polymers which are
resistant to strong acids and alkalis.
The column dimensions are also important and a
length: diameter ratio of 20: 1to 100: 1 for higher
efficiency can be used.
2. Type of ion exchange resin:
Type of ions cations (or) anions
nature of ions Strong (or) weak
Efficiency of the resin It is measured by ion
exchange capacity
18. Ion exchange capacity:
It is the total ion exchange capacity in terms of
the exchangable functional groups expressed as milli
equivalents per gram of the ion exchange resin.
Particle size of the resin Porous, Pellicular
Amount of the cross linking agent
Which decides swelling of the resin
3. PACKING OF THE COLUMN:
Wet packing method is used.
Resin + Mobile phase – packing in the column
uniformly.
m.Eq / g =1000 / eq.wt
19. 4. MOBILE PHASE:
Organic solvents are less useful and they are not used at
all.
Only strengths of acids, alkalies and buffers are used as
eluting solvents.
E.g. 0.1N HCL, 1N NaOH, Phosphate buffer,Acetate
buffer, Borate buffer, phthalate buffer, etc
5. DEVELOPMENT OF THE CHROMATOGRAM
AND ELUTION
After introduction of the sample, development of the
chromatogram is done by using different mobile phases.
As, mentioned earlier, organic solvents are less useful and
only acids, alkalis and buffers of different pH are used.
20. There are two elution technique:
1. Isocratic elution
2.Gradient elution
Isocratic elution:
Same solvent composition is used. i.e., same
solvent of acid or alkali or buffer.
Gradient elution:
In gradient elution technique, initially less acidic or
basic character is used followed by increasing the
acidity or basicity of the mobile phase.
this elution technique is usually used for complex
mixtures. The different fractions of the eluent is
collected volume wise or time wise and analysed.
21.
22.
23. ANALYTES OF THE ELUTE:
several methods of analysis can be used which
depends up on the nature & the quantity of the sample.
1. Spectrophotometric method
2. Polarographic method
3. Conductometric method
4. Amperometric method
5. Flame photometric method
6. Radio chemical methods
Geiger muller counter
ionization chamber method.
after analyzing similar fraction are mixed in order
to get pure ions or compound of each type.
24. REGENERATION OF THE ION EXCHANGE
RESIN:
o
o
o Regeneration makes the used ion exchange resin to be as
efficient as a virgin resin.
o Regeneration refers to the replacement of the
exchangable cations or anions present in the original
resin.
Hence regeneration of the cation exchange resin is done
by the charging the column with strong acid like HCl
acid.
Regeneration of anion exchange resin is done by using
strong alkali like sodium hydroxide or potassium
hydroxide.
26. FACTORS AFFECTING ION EXCHANGE
SEPARATIONS:
1. Nature and properties of ion exchange resins.
2. Nature of exchanging ions.
1. Nature and properties of ion exchange resins:
Cross linking and swelling is important factor
which depends on the proportion of cross linking agent
is and polystrene.
when more cross linking agent is present, they are
more rigid, but swells less.
When swelling is less, separation of different sizes
is difficult as they cannot pass through the pores
present and it becomes selective to ions of different
sizes.
27. 2. Nature of exchanging ions:
a. Valency of ions
b. Size of ions
c. Polarizability
d. Concentration of solution
e. Con. & charge of ions
28. CONCENTRATION AND CHARGE OF IONS:
If resin has higher +ve charge and solution has lower
+ve charge, exchange is favoured at higher
concentration.
If the resin has lower +ve charge and solution has
high +ve charge, then exchange is favoured at low
concentration.
29. APPLICATIONS:
Conversion from one salt to other e.g. we can
prepare tetra propyl ammonium hydroxide from a
tetra propyl salt of some other anion.
Household (laundry detergents and water filters) to
produce soft water
Ion exchange is used to prepare de-ionized water
Separate and purify metals
Dealkalization
Analysis and purification of immunoglobulin's
Separation of inorganic ions
