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
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
HPLC is a High Performance liquid Chromatography.
High Pressure Liquid Chromatography.
High Priced Liquid Chromatography.
It is column chromatography.
It is Liquid Chromatography.
It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile compound.
It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
It is having a high resolution and separation capacity.
Introduction
History
Elecrophoresis
Principle
Types of electrophoresis
Application
Conclusion
Reference
When a potential difference is applied between the two electrodes in a colloidal solution, It has been observed that the colloidal particles are carried to either the positive or negative electrode.
In other words , they behave as if they are electrically charged w.r.t. the dispersion medium. This phenomenon is known as electrophoresis.
Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations or anions.
Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.
HPLC is a High Performance liquid Chromatography.
High Pressure Liquid Chromatography.
High Priced Liquid Chromatography.
It is column chromatography.
It is Liquid Chromatography.
It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile compound.
It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
It is having a high resolution and separation capacity.
Introduction
History
Elecrophoresis
Principle
Types of electrophoresis
Application
Conclusion
Reference
When a potential difference is applied between the two electrodes in a colloidal solution, It has been observed that the colloidal particles are carried to either the positive or negative electrode.
In other words , they behave as if they are electrically charged w.r.t. the dispersion medium. This phenomenon is known as electrophoresis.
Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations or anions.
Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.
In gas chromatography, the sample is first converted to the vapor state or volatile compound is
used which are injected into heated port along with a carrier gas. Analyte in the vapor state
distributes between the stationary phase and the carrier gas. As the analyte elute from column
signal is displayed in the form of chromatogram. Measurements of GC are rapid and convenient.
Retention times are used for qualitative identification. Peak areas are used for quantitative
measurements.
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Nucleophilic Addition of carbonyl compounds.pptxSSR02
Nucleophilic addition is the most important reaction of carbonyls. Not just aldehydes and ketones, but also carboxylic acid derivatives in general.
Carbonyls undergo addition reactions with a large range of nucleophiles.
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Exposé invité Journées Nationales du GDR GPL 2024
2. CHROMATOGRAPHY
• Chromatography is an analytical technique where in a
sample mixture under test is separated into different
components based on difference in their affinity for a
stationery phase and mobile phase.
3.
4.
5. GAS LIQUID
CHROMATOGRAPHY(GLC)
• In GLC the mobile phase is an unreactive gas , such as
nitrogen(Carrier gas).
• The stationery phase is a non
volatile liquid held on a finely
divided inert solid support.
6. PRINCIPLE
• Gas liquid chromatography runs on the principle of partition.
• In GLC , components of the gaseous samples are separated due
to partition between a gaseous mobile phase and a liquid
stationery phase held in a column.
7.
8.
9. The mobile phase
• An inert gas such as He or N2
-Function is to transport sample vapours through columns.
-No chemical interaction with sample.
• Typical parameters
-column inlet pressure : 10-50psi
-flow rate 25-50mL/min
• Precise control of carrier gas flow rate is crtical to obtain
reproducible retention times.
10. Sample injection
Sample is injected using a syringe into a hot carrier gas(mobile
gas)
-High temperature( at least 50 ͦC above B.P of sample)
causes vaporization of sample.
- introduced a narrow plug of sample vapour onto the
column.
Various designs
-for packed columns , inject 1 to 5 microliters of sample.
-for capillary columns , a split valve is used to introduce a
small fraction sample into the column .
11.
12.
13.
14.
15.
16.
17. Detectors
• Generate an electrical signal proportional tp solute concentration
or mass flow rate.
Ideal characteristics :
• High sensitivity
• Rapid response time
• Applicable to wide range of samples
• Easy to use
• Stable & predictable response
18.
19.
20.
21.
22.
23.
24.
25.
26. RECORDER
• Recorder is device that draws the chromatogram that results
from a chromatographic process on to chart paper.
• The time scale of the chart movement normally ranges from
about 1cm/ second to 1cm / hour
27. Advantages of GLC
• Both qualitative and quantitative analysis are possible.
• Instrument is simple , time of analysis is short.
• High sensitivity
• This method is applicable for 60% of organic compounds
• Very small sample sizes can be used
• Analysis can be highly accurate and precise.
28. FACTORS AFFECTING SEPERATION
• Particle size and surface area
• Carrier gas flow rate
• Type and amount of stationery phase
• Column length
• Column diameter
• Column temperature
29. CONCLUSION
• Gas chromatography (GC) is a common type of
chromatography used in analytical chemistry for
separating and analyzing compounds that can be vaporized
without decomposition.
• The sample being measured is injected into the carrier gas
using a syringe and instantly vaporizes (turns into gas
form).
• The gases that make up the sample separate out as they
move along the column which contains the stationary
phase
30. REFERENCES
• Introduction and principle of GLC , by Dr.Vishnu Vrardhan
Reddy Pulimi , www.slideshare.net
• Gas liquid chromatography by Srinivas Nandyala ,
m.athorstream.com