ION EXCHANGE CHROMATOGRAPHY
ByM.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
ION EXCHANGE CHROMATOGRAPHY
ByM.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 gives you thorough knowledge about the IR Spectroscopy. This include basic principle, type of vibrations, factors influencing vibrational frequency, instrumentation and applications of IR Spectroscopy. This is the most widely used technique for identifying unknown functional group depending on the vibrational frequency.
In this slide contains types, working principle, factors affecting, advantage and disadvantage of paper electrophoresis.
Presented by: G.Sai Swetha. (Department of pharmacology),
RIPER, anantapur.
This presentation gives you thorough knowledge about the IR Spectroscopy. This include basic principle, type of vibrations, factors influencing vibrational frequency, instrumentation and applications of IR Spectroscopy. This is the most widely used technique for identifying unknown functional group depending on the vibrational frequency.
In this slide contains types, working principle, factors affecting, advantage and disadvantage of paper electrophoresis.
Presented by: G.Sai Swetha. (Department of pharmacology),
RIPER, anantapur.
TLC-Introduction, Principle, Procedure, and Applications.
Paper Chromatography-Introduction, Principle, Procedure, and Applications.
Column Chromatography-Introduction, Principle, Procedure, and Applications.
Chromatography is a bioanalytical technique used for separation of analytes into pure components. Biomolecules such as amino acids, proteins and carbohydrates can be purified by different chromatographic methods.
Chromatography : A seperation techniqueSHIVANEE VYAS
Chromatography is a method of seperating mixture of components into individual components through equlibrium distribution between two phases.
Each chromatographic method essentially consists of 2 phases a staionary phase and a mobile phase.
Stationary phase : solid or liquid
Mobile phase : liquid or gas
Cyclohexane exists in different conformations viz chair, boat, twist boat and half chair. These conformations possess different energies. Therefore they differ in energy.
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
We all have good and bad thoughts from time to time and situation to situation. We are bombarded daily with spiraling thoughts(both negative and positive) creating all-consuming feel , making us difficult to manage with associated suffering. Good thoughts are like our Mob Signal (Positive thought) amidst noise(negative thought) in the atmosphere. Negative thoughts like noise outweigh positive thoughts. These thoughts often create unwanted confusion, trouble, stress and frustration in our mind as well as chaos in our physical world. Negative thoughts are also known as “distorted thinking”.
How to Create Map Views in the Odoo 17 ERPCeline George
The map views are useful for providing a geographical representation of data. They allow users to visualize and analyze the data in a more intuitive manner.
MARUTI SUZUKI- A Successful Joint Venture in India.pptx
Paper chromatography
1. Techniques of separation
Module : Chromatography
Topic : Paper Chromatography
By
Dr. M. T. Bachute
Dept. of Chemistry
K.B.P. Mahavidyalaya, Pandharpur
2. Paper Chromatography
Paper chromatography is a type of Planar
chromatography in which paper is the support
for stationary phase.
It is a partition chromatography.
Stationary Phase : Liquid, deposited in network
of cellulose fibers.
Mobile Phase : Liquid, solvent(single or
mixture)
It is a liquid – liquid chromatography
3. Principle
It is based on the principle of partition of a compound
between two liquid phases.
The water hold in cellulose fibers of the paper acts as the
stationary phase and acts as one of the solvents (liquids).
When a non–aqueous mobile phase moves along the paper,
the components of the loaded mixture distribute
themselves between two phases in the ratio characteristic
of their distribution coefficients.
The component which is more soluble in stationary phase
moves slowly while the component which is more soluble in
mobile phase moves fast.
As a result of this differential movement, components get
separated.
Distribution coefficient = (solute in stationary phase) ÷ (solute in mobile phase)
4. Important Aspects of Chromatography
1. Filter Paper
2. Solvent System
3. Spraying reagents
5. Filter Paper
• Usually Whatman paper No. 1 is used.
Characters : Uniform flow rate
Grain size and texture
• Some times the papers are acid
washed to remove traces of
impurities which may affect
the analysis.
Strip of whatman No. 1 paper
6. Solvent system
Solvent is prepared by saturating organic solvent
with water.
But several organic liquids incorporate only small
amount of water. In such binary solvent system
polar components of the mixture fail to
separate.
In such cases a third liquid like acid(HCl, AcOH,
HCOOH) or base(NH3 or pyridine) or a
complexing agent is often added to retain more
water in organic solvents.
Hence, generally three component solvent
systems are used
7. Some important solvent systems
Compounds Solvent system Spraying reagent
1. Amino acids Butanol: Pyridine:Water
(33 :33 :33)
Ninhydrin
Methanol:Pyridine:Water
(25 : 12 :63)
Butanol : AcOH : Water
(40 : 19 50)
Phenol : Water
(80 : 20)
2. Carbhydrates(mono , di) Butanol : AcOH : Water
Butanol:
Pyridine:Water:AcOH
(8:8:4:1)
Ammoniacal AgNO3
Alkaline KMnO4
3. Chlorophils and
carotenoids
Propanol : Pet. Ether
Chloroform : Pet. ether
Self
4. Metal ions Ethanol: 5M HCl (90 : 10) Rubeanic acid, NH3
8. Spraying reagents
• Coloured components do not require spraying
reagent, they are visible.
• Colourless components are located by
spraying with specific reagents. These
reagents chemically react with the
components and develop colour in the region
where they are present.
9. Methodology
1. Sample loading
Preparation of sample solution :
1-2% solution is prepared in suitable
solvent.
A reference line at 2cm . Distance
from the edge of the paper is drawn
and points of application are marked.
The solution of mixture under test is
spotted either as a fine line or fine
spot. Spotting is repeated twice or
thrice. Reference Line .
10. Methodology
2. Developing
Saturation of the paper strip: The sample loaded strip is
hanged for some time in the chromatographic jar
containing the solvent but without dipping in the
solvent.
The strip is developed by running the solvent through the
paper.
Depending on the direction of flow, there are two types
paper chromatography.
A. Ascending paper chromatography
B. Descending paper chromatography
11. Ascending P. C.
Solvent moves upward(ascends) from the
bottom to top by capillary action. The paper
strip is placed vertically in the solvent keeping
the reference line above the solvent level.
Pencil
Filter
Paper
Ink
Mark
Tape – Label with marker
12. Descending P. C.
• Solvent moves downward(descends) from the
top to bottom. The paper strip is hanged
vertically downward from the trough
containing solvent, keeping the spotted end
at the top.
The flow of solvent is faster than that in
ascending P.C.
But A.P.C. is preferred than D.P.C. due to
simplicity of set up.
13. Radial or Circular chromatography
The plane of paper is kept horizontal and solvent
moves laterally(toward the periphery) from
the center.
14. Methodology
3. Spraying : After movement of the solvent to
expected distance(15 to 18 cm), paper is
removed, dried and sprayed with suitable
reagent.
16. Rate of Flow : Rf value
Rf value : It is the ratio of distance travelled by
the solute to the distance travelled by
the solvent front. i.e.
Distance moved by solute
Rf =
Distance moved by the solvent front
Rf value …….
• characterises the distance moved by the solute in the direction of solvent
flow
• under standard conditions it closely reflects the distribution coefficient
and it is characteristic of that compound
17. Rf value
Factors affecting Rf value
1. Quality and dimension of the paper.
2. Solvent system, its flow rate and direction of
flow.
3. Temperature of the environment
4. Size of the vessel in which chromatogram is
developed.
If these factors are kept constant, Rf values of
known and unknown compounds can be
compared.
18. Applications of Paper Chromatography
1. Separation of amino acids, peptides,
alkaloids, sugars, lipids etc in biological
samples.
2. To limited extent as a preparative method.
3. To study the structure and amino acid
composition of proteins
4. Analysis of blood, hemoglobin, urine etc
which is of great diagnostic value.
19. Advantages
1. Method is simple, inexpensive and sensitive.
2. Separations can be carried out under
ordinary lab. conditions.
20. Disadvantages
1. Time required is large.
2. It can not be used as a preparative method.
3. Heating or corrosive reagents can not be
used for detection.
21. Advantages of TLC over PC
1. Versatility in the use of stationary phase.
It offers choice between wide range of adsorbents to suite the
separation of variety of components.
2. Adsorbent layer of variable thickness(upto 5mm)can be obtained.
Higher sample load is possible.(higher load on paper not possible)
3. Method is very rapid & most of the separations can be completed
within an hour.
(PC requires 18 to 24 hrs.)
4. Separated spots are very compact, hence detection at lower
concentration is possible.
(In PC spots are diffused)
5. Higher temp. and corrosive sprays can be used to detect unknown
solutes.( higher temp and corrosive sprays can not be used with
paper.)
6. Since it is simple, rapid and inexpensive, it can be used to check
purity of synthesised or isolated chemicals.
22. Disadvantages of TLC
1. Rf values are not reproducible. Rf value is
affected by thickness of the layer below
0.2mm and also by uneven thickness of the
layer.